CN107406422B - Pyrazole derivatives as sGC stimulators - Google Patents

Abstract

The compounds of the invention are useful as stimulators of sGC, especially NO-independent, heme-dependent stimulators. These compounds are also useful for treating, preventing or managing the various disorders disclosed herein.

Description

Pyrazole derivatives as sGC stimulators

Cross Reference to Related Applications

This application claims priority from U.S. provisional application No. 62/051,557 filed on 9/17 of 2014 and U.S. provisional application No. 62/204,710 filed on 8/13 of 2015. The entire contents of each of these applications are incorporated herein by reference.

Technical Field

The present application relates to Stimulators/activators of soluble guanylate cyclase (sGC) (Stimulators), pharmaceutical formulations containing them and their use, alone or in combination with one or more other drugs, for the treatment and/or prevention of various diseases, where an increase in the concentration of Nitric Oxide (NO) or increasing the concentration of cyclic guanylic acid (cGMP) is desirable.

Background

Soluble guanylate cyclase (sGC) is the major receptor for Nitric Oxide (NO) in vivo. sGC can be activated by both NO-dependent and NO-independent mechanisms. In response to this activation, sGC converts GTP to the second messenger cyclic guanosine monophosphate (cGMP). The increase in cGMP levels in turn modulates the activity of downstream effector molecules, including protein kinases, Phosphodiesterases (PDEs), and ion channels.

In vivo, NO is synthesized from arginine and oxygen by the action of various Nitric Oxide Synthases (NOs) and by successive reduction reactions of inorganic nitrate. There are three currently identified subtypes of NOS: inducible NOS (iNOS or NOSII) found in activated macrophages; constitutive neuronal NOS (nNOS or NOSI) involved in neurotransmission and long-range potentiation; and constitutive endothelial NOS (eNOS or nosii) that regulates smooth muscle relaxation and blood pressure.

Experimental and clinical evidence suggests that a decrease in endogenous NO bioavailability and/or responsiveness may contribute to the development of cardiovascular, endothelial, renal and liver disease, as well as erectile dysfunction and other sexual disorders (e.g., female sexual disorder or vaginal atrophy). In particular, NO signaling pathways are altered in a variety of cardiovascular diseases such as systemic and pulmonary hypertension, heart failure, angina, stroke, thrombosis and other thromboembolic disorders, peripheral arterial disease, liver, lung or kidney fibrosis, and atherosclerosis.

sGC stimulators are also useful in the treatment of lipid-related diseases such as dyslipidemia, hypercholesterolemia, hypertriglyceridemia, sitosterolemia, fatty liver disease and hepatitis.

Pulmonary Hypertension (PH) is a disease characterized by a sustained rise in blood pressure in the pulmonary vessels (pulmonary arteries, pulmonary veins and pulmonary capillaries) that leads to right heart hypertrophy, ultimately leading to right heart failure and death. At PH, NO and other vasodilators such as prostacyclin have reduced biological activity, while the production of endogenous vasoconstrictors such as endothelin is increased, resulting in excessive pulmonary vasoconstriction. sGC stimulators have been used to treat PH because they promote smooth muscle relaxation, leading to vasodilation.

Treatment with NO-independent sGC activators also promotes smooth muscle relaxation in the corpus cavernosum of the penis in healthy rabbits, rats and humans, leading to penile erection, suggesting that sGC stimulators may be useful in the treatment of erectile dysfunction.

NO-independent, heme-dependent sGC activators such as those disclosed herein have several important distinguishing features: including strong dependence of its activity on the presence of reduced heme prosthetic groups, strong synergistic enzymatic activity in combination with NO, and NO-independent stimulation of cGMP synthesis by direct stimulation of sGC. The benzylic indazole compound YC-1 was the first identified sGC stimulator. More sGC stimulators with stronger effect and higher selectivity have also been developed. These compounds have shown anti-aggregation, anti-proliferative and vasodilatory effects.

Since compounds that stimulate sGC in an NO-independent manner have significant advantages over other current alternative therapies, the development of novel sGC stimulators is essential. They have potential in the prevention, management and treatment of diseases such as pulmonary hypertension, arterial hypertension, heart failure, atherosclerosis, inflammation, thrombosis, renal fibrosis and failure, liver cirrhosis, pulmonary fibrosis, erectile dysfunction, female sexual arousal disorder and vaginal atrophy and other cardiovascular diseases; and has open prospects in the prevention, management and treatment of lipid-related diseases.

Disclosure of Invention

The present invention relates to compounds useful as sGC Stimulators/activators (Stimulators) or pharmaceutically acceptable salts thereof. The compounds of the invention are described in table 1A or table 1B.

TABLE IA

TABLE IB

The present invention also relates to pharmaceutical compositions comprising a compound from table 1A or table 1B, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier. The invention also relates to a pharmaceutical preparation or dosage form comprising the pharmaceutical composition.

The present invention also provides a method of treating or preventing a disease, health condition or disorder in a subject in need thereof, comprising administering an effective amount of a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof, alone or in combination; wherein the disease, health condition or disorder comprises a peripheral, pulmonary, hepatic, renal, cardiac or cerebrovascular/endothelial disease or disorder, a urogenital or sexual disorder or disorder, a thromboembolic disease, a fibrotic disease, a pulmonary or respiratory disease, a renal or hepatic disorder, an ocular disorder, a hearing disorder, a CNS disorder, a circulatory disorder, a topical or dermal disorder, a metabolic disorder, atherosclerosis, wound healing or a lipid-related disorder that benefits from sGC stimulation or an increase in the concentration of NO or cGMP.

Detailed description of the invention

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. The present invention is not limited to the methods and materials described herein, but includes any methods and materials similar or equivalent to those described herein that can be used to practice the present invention. In the event that one or more of the incorporated literature references, patents, or similar materials differ or contradict the present application, including but not limited to defined terms, term usage, described techniques, or the like, the present application controls.

Definitions and general terms

For the purposes of the present invention, the periodic Table of the elements (CAS version) and the 75 th edition of the Handbook of Chemistry and Physics (75 dbook of Chemistry and Physics) are used^thEd) to identify the chemical element. Furthermore, the general principles of Organic Chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books (University Science Books), sudox (sautalito): 1999 and "March's Advanced Organic Chemistry", 5 th edition, Smith, M.B. and March, J., John Wiley International publishing Co., John &Sons), New York (New York): 2001, the entire contents of which are incorporated herein by reference.

Compounds such as the compounds of table 1A or table 1B or other compounds disclosed herein may exist in their free form (e.g., amorphous form, or crystalline form or polymorph). Under certain conditions, the compounds may also form co-forms. As used herein, the term co-form is synonymous with the term multicomponent crystalline form. When one component of the co-form apparently transfers protons to the other component, the resulting co-form is referred to as a "salt". Salt formation is determined by how much the pKa of the ligands forming the mixture differs. For the purposes of the present invention, compounds include pharmaceutically acceptable salts even if the term "pharmaceutically acceptable salt" is not explicitly indicated.

Unless only one isomer is specifically depicted or named, the structures described herein are also intended to include all stereoisomeric (e.g., enantiomeric, diastereomeric, atropisomeric and cis-trans isomer) forms of the structure; for example, the R and S configurations for each asymmetric center, the Ra and Sa configurations for each asymmetric axis, (Z) and (E) double bond configurations, and cis and trans conformers. Thus, single stereochemical isomers as well as racemates of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers and cis-trans isomers (double bonds or conformations), are within the scope of the present invention. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are also within the scope of the invention. By way of example, the substituents are as follows:

Where R may be hydrogen, two compounds shown below will be included:

the invention also includes isotopically-labeled compounds which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. All isotopes of any particular atom or element and uses thereof are contemplated as being within the scope of the compounds of the present invention. Exemplary isotopes that can be substituted in compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, for example, each of²H，³H，¹¹C，¹³C，¹⁴C，¹³N，¹⁵N，¹⁵O，¹⁷O，¹⁸O，³²P，³³P，³⁵S，¹⁸F，³⁶Cl，¹²³I and¹²⁵I. certain isotopic labels of the present inventionOf (e.g. with)³H and¹⁴c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e. by tritiation)³H) And carbon-14 (i.e.¹⁴C) Isotopes are useful for their ease of preparation and detection. In addition, heavier isotopes such as deuterium (i.e., deuterium) are used²H) Substitution may provide certain therapeutic advantages due to its higher metabolic stability (e.g., longer in vivo half-life or lower dosage requirements) and may therefore be preferred in some circumstances. Positron emitting isotopes such as¹⁵O，¹³N，¹¹C and¹⁸f can be used in Positron Emission Tomography (PET) studies to examine the receptor occupancy of substrates. Isotopically labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

The compounds of the present invention are defined herein by their chemical structure and/or chemical name. When the chemical structure and chemical name of a compound are referred to at the same time, and the chemical structure and chemical name conflict, the chemical structure controls.

Compound (I)

The compounds of the present invention are selected from those described in table 1A or table 1B.

TABLE IA

TABLE IB

Process for the preparation of compounds

The compounds of the present invention may be prepared according to the schemes and examples described and illustrated below. Unless otherwise indicated, starting materials and various intermediates can be obtained from commercial sources, prepared from commercially available compounds or prepared using well known synthetic methods.

The general synthetic methods for the compounds of the present invention are described below. The synthesis schemes are given as examples and do not limit the scope of the invention in any way.

General method A

Step 1:

diketone enolate formation: to a solution of ketone a in THF cooled to-78 ℃, LiHMDS (e.g., 0.9 eq, 1.0M in toluene) was added dropwise via syringe. The reaction was warmed to 0 ℃ and diethyl oxalate (1.2 eq) was added. At this point, the reaction is warmed to room temperature and stirred at that temperature until the reaction is complete (e.g., using TLC or LC/MS analysis). Once the reaction is complete (reaction time is typically 45 minutes), the product diketoenolate B is used "directly" in step 2, i.e. the cyclisation step, without any further purification.

Step 2:

pyrazole formation: diketoenolate B was diluted with ethanol and HCl (e.g., 3 equivalents, 1.25M in ethanol) and aryl hydrazine hydrate (e.g., 1.15 equivalents) were added continuously. The reaction mixture is heated to 70 ℃ and stirred at this temperature until the cyclization reaction is complete (e.g., by LC/MS analysis, typically 30 minutes). Once complete, the reaction mixture is carefully treated with solid sodium bicarbonate (e.g., 4 equivalents) and diluted with dichloromethane and water. The layers were separated and the aqueous layer was further diluted with water and then extracted with dichloromethane (3 ×). The combined organic layers were washed with brineWashing with MgSO 2₄Dried, filtered and concentrated in vacuo. Then pass through SiO using an appropriate gradient of EtOAc/hexanes solution₂And (4) carrying out chromatography purification to obtain pyrazole C.

And step 3:

amidine formation:to NH cooled to 0 DEG C₄AlMe was added dropwise via syringe to a suspension of Cl (e.g., 5 equivalents) in toluene₃(e.g., 5 equivalents, 2.0M in toluene). The reaction was warmed to room temperature and stirred at that temperature until no more bubbles were observed. Pyrazole C is added to the reaction mixture in one portion, heated to 110 ℃, and stirred at that temperature until the reaction is judged to be complete (e.g., using TLC or LC/MS analysis). Once complete, the reaction was cooled, treated with excess methanol, and stirred vigorously at room temperature for 1 hour. The viscous slurry was filtered and the resulting solid filter cake was washed with methanol. The filtrate was concentrated in vacuo and the resulting solid was resuspended in ethyl acetate: isopropanol in a solvent mixture in a volume ratio of 5: 1. The reaction was further treated with saturated sodium carbonate solution, stirred for 10 minutes, and then the layers were separated. The aqueous layer was washed with ethyl acetate: a solvent mixture of isopropanol ═ 5:1 was extracted three times and the combined organic layers were washed with brine. The organic layer was further treated with MgSO ₄Drying, filtering and removing the solvent in vacuum. The product, amidine D, was used directly in the next step without further purification.

And 4, step 4:

pyrimidinone formation:amidine D was suspended in ethanol and stirred vigorously at 23 ℃ to promote complete solvation. The reaction was further treated with sodium 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (e.g., 3 equivalents) and the flask was equipped with a reflux condenser. The reaction was placed in a pre-heated oil bath maintained at 90 ℃ and stirred until complete consumption of starting material was observed by LC/MS (reaction time typically 1 hour). The reaction was cooled to 23 ℃, and the reaction mixture was acidified with HCl (e.g., 3 equivalents, 1.25M EtOH solution). The mixture was stirred for 30 minutes and most of the solvent was removed in vacuo. The contents were resuspended in ether and water (1:1 mixture) and the resulting slurry was stirred for 20 minutes. Will be provided withThe suspension was filtered under vacuum and the solid cake was washed with additional water and ether and dried under high vacuum overnight. The pyrimidinone E obtained is used directly in the subsequent step without further purification.

General method B

The amino nucleophile (3 equiv.), triethylamine (10 equiv.) and intermediate-1A (1 equiv.) were stirred in dioxane and water (2:1 ratio) at 90 ℃ until complete consumption of starting material was observed by LC/MS. The solution was diluted with 1N aqueous hydrochloric acid and dichloromethane. The layers were then separated and the aqueous layer was extracted with dichloromethane. The organic layers were combined, dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification to give the desired product.

General method C

To a solution of intermediate-2 (which was described in the previously published patent application WO2012/3405a 1; 1 eq) and a carboxylic acid (1.1 eq) in N, N-dimethylformamide was added successively triethylamine (4 eq) and a solution of 50% propylphosphonic anhydride (T3P, 1.4 eq) in ethyl acetate. The reaction was heated to 80 ℃ for 24 hours and then the reaction was diluted with water and 1N hydrochloric acid solution. Extracted with dichloromethane and then ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Purification to give the desired product.

Pharmaceutically acceptable salts of the invention

In all cases described herein, the term "compound" also includes pharmaceutically acceptable salts of the compounds, whether or not the phrase "pharmaceutically acceptable salts" is actually used. The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds of table 1A or table 1B. Pharmaceutically acceptable salts of the compounds of table 1A or table 1B are used in medicine. However, salts that are not pharmaceutically acceptable may be used to prepare the compounds of table 1A or table 1B or pharmaceutically acceptable salts thereof. Pharmaceutically acceptable salts may be contemplated to include another molecule, such as an acetate ion, succinate ion or other counter ion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, pharmaceutically acceptable salts may have more than one charged atom in their structure. Embodiments in which the plurality of charged atoms are part of a pharmaceutically acceptable salt may have a plurality of counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

Pharmaceutically acceptable salts of the compounds described herein include those derived from compounds having inorganic, organic or base acids. In some embodiments, the salts may be prepared in situ during the final isolation and purification of the compounds. In other embodiments, the salts may be prepared in a separate synthetic step from the free form of the compound.

When the compounds of table 1A or table 1B are acidic or contain sufficiently acidic bioisosteres, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Specific examples include ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N, N-benzhydrylvinyldiamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the compounds of table 1BA or table 1B are basic or contain sufficiently basic bioisosteres, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. These acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Specific examples include citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid. Other exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)) salts.

The above pharmaceutically acceptable Salts and other typical pharmaceutically acceptable Salts are prepared by Berg et al in "pharmaceutically acceptable Salts (Pharmaceutical Salts)," journal of Pharmaceutical science (j.pharm.sci.), 1977: 66: 1-19, which are incorporated herein by reference in their entirety.

In addition to the compounds described herein, their pharmaceutically acceptable salts may also be used in compositions to treat or prevent the conditions indicated herein.

In all cases described herein, the term "compound" also includes pharmaceutically acceptable salts of the compounds, whether or not the phrase "pharmaceutically acceptable salts" is actually used.

Pharmaceutical compositions and methods of administration

The compounds disclosed herein and pharmaceutically acceptable salts thereof may be formulated as pharmaceutical compositions or "formulations".

A typical formulation is prepared by mixing a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof, with a carrier, diluent, or excipient. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient used will depend on the means and purpose for which the compound of table 1A or table 1B is formulated. The choice is generally based on the skilled artisan to consider safe (GRAS-generally regarded as safe) solvents for administration to mammals. Generally, safe solvents are non-toxic aqueous solvents, such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG400, PEG300), and the like, and mixtures thereof. The formulations may also include other types of excipients, such as one or more buffering agents, stabilizing agents, anti-adherents, surfactants, wetting agents, lubricants, emulsifiers, binders, suspending agents, disintegrants, fillers, adsorbents, films (e.g., enteric or sustained release) agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents, and other known additives to provide a finished presentation of a drug (i.e., a compound of table 1A or table 1B or pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (i.e., a drug).

The formulations can be prepared using conventional dissolution and mixing procedures. For example, the starting drug (i.e., a compound of table 1A or table 1B, a pharmaceutically acceptable salt thereof, or a stable form of the compound, e.g., a complex with a cyclodextrin derivative or other known complexing agent) is dissolved in a suitable solvent in the presence of one or more of the above excipients. The compound of the desired purity may optionally be admixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers in the form of a lyophilized preparation, a milled powder or an aqueous solution. The formulations may be prepared by mixing at ambient temperature, at an appropriate pH and at the required purity, with a physiologically acceptable carrier. The pH of the formulation depends primarily on the particular use and concentration of the compound, but can range from about 3 to about 8. When the agent described herein is a solid amorphous dispersion formed by a solvent process, the additive may be added directly to the spray-dried solution as the mixture is formed, for example by dissolving or suspending the additive in solution as a slurry which may then be spray-dried. Alternatively, additives may be added after the spray drying process to help form the final formulated product.

The compounds of table 1A or table 1B, or pharmaceutically acceptable salts thereof, are generally formulated in a pharmaceutical dosage form that allows for easy control of the dosage of the drug and that allows the patient to be matched for prescribed use. Such pharmaceutical dosage forms prepared with the compounds of table 1A or table 1B, or pharmaceutically acceptable salts thereof, can be used for a variety of routes and types of administration. Since different medical conditions require different routes of administration, the same compound may exist in multiple dosage forms.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain from about 1 to 1000mg of the active substance, together with a suitable and convenient amount of carrier material, which may be from about 5 to about 95% by weight of the total composition (w: w). The pharmaceutical composition may be prepared to provide an amount that is easily measured. For example, an aqueous solution for intravenous infusion may contain from about 3 to 500 μ g of active ingredient per mL of solution so as to enable infusion of a suitable volume at a rate of about 30 mL/hr. As a general proposition, the initial pharmaceutically effective amount of inhibitor administered will be in the range of about 0.01-100mg/kg dose, i.e., about 0.1 to 20mg/kg patient body weight per day, with the initial compound typically used in the range of 0.3-15 mg/kg/day.

The term "therapeutically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. A therapeutically or pharmaceutically effective amount of a compound to be administered will be governed by such considerations and is the minimum amount required to ameliorate, cure or treat the disease or disorder or one or more symptoms thereof.

Pharmaceutical compositions of the compounds of table 1A or table 1B will be formulated, dosed and administered in amounts, concentrations, schedules, courses of treatment, carriers and routes of administration consistent with good medical practice. Factors considered herein include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners, such as the age, weight, and response of the individual patient.

The term "prophylactically effective amount" refers to an amount effective in either preventing or substantially reducing the chance of, or lessening the severity of, a disease or disorder prior to the development of the disease or disorder, or in one or more symptoms thereof before the symptoms thereof manifest. Broadly, preventive measures are divided into primary prevention (to prevent the onset of disease) and secondary prevention (disease has already occurred and thus protects the patient from worsening the process).

Acceptable diluents, carriers, excipients, and stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins, for example serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelators, such as EDTA; sugars, for example sucrose, mannitol, trapolysaccharide or sorbitol; salt-forming counterions, such as sodium; metal complexes (for example Zn-protein complex)Compound(s); and/or nonionic surfactants, e.g. tweens (tweens)^TM) Pluronic (pluronic) ^TM) Or polyethylene glycol (PEG). The active pharmaceutical ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively; in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in emulsions. These techniques are disclosed in Remington's, Remington, 2005: pharmaceutical Sciences and practices (The Science and Practice of Pharmacy), 21 st edition, Philadelphia University of The Sciences in Philadelphia (hereinafter "Remington's").

A "controlled drug delivery system" provides a drug to the body in a precisely controlled manner appropriate to the drug and the condition being treated. The primary objective is to achieve therapeutic drug concentrations at the site of action within the desired time. The term "controlled release" is generally used to refer to a variety of methods for modifying the release of a drug from a dosage form. The term includes formulations labeled "extended release", "delayed release", "modified release" or "sustained release". In general, controlled release of the agents described herein can be provided through the use of a wide variety of polymeric carriers and controlled release systems, including erodible and non-erodible matrices, osmotic control devices, various storage devices, enteric coatings and multiparticulate control devices.

"sustained release formulations" are the most common application of controlled release. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the compound, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, nondegradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D- (-) -3-hydroxybutyric acid.

"immediate release formulations" may also be prepared. The purpose of these formulations is to get the drug into the blood and the site of action as quickly as possible. For example, for rapid dissolution, most tablets are designed to undergo rapid disintegration into granules and subsequent disaggregation into fine particles. This provides a greater surface area exposed to the dissolution medium, resulting in a faster dissolution rate.

The agents described herein may be incorporated into erodible or non-erodible polymeric matrix controlled release devices. By erodible matrix is meant an agent that is water erodible or water swellable or water soluble, i.e., is erodible or swellable or soluble in pure water, or requires the presence of an acid or base to ionize the polymer matrix sufficiently to cause erosion or dissolution. Upon contact with the aqueous environment of use, the erodible polymeric matrix absorbs water and forms a water-swellable gel or matrix that entraps the agents described herein. The water-swellable matrix gradually erodes, swells, disintegrates or dissolves in the environment of use, thereby controlling the release of the compounds described herein to the environment of use. One component of the water-swellable matrix is a water-swellable, erodible or soluble polymer, which can be generally described as an osmopolymer, hydrogel or water-swellable polymer. Such polymers may be linear, branched or crosslinked. The polymer may be a homopolymer or a copolymer. In certain embodiments, they may be synthetic polymers derived from vinyl, acrylate, methacrylate, urethane, ester and oxide monomers. In other embodiments, they may be derivatives of naturally occurring polymers, such as polysaccharides (e.g., chitin, chitosan, dextran and amylopectin; agar, gum arabic, karaya gum, locust bean gum, tragacanth gum, carrageenan, ghatti gum, guar gum, xanthan gum and scleroglucan), starches (e.g., dextrin and maltodextrin), hydrocolloids (e.g., pectin), phospholipids (e.g., lecithin), alginates (e.g., ammonium, sodium, potassium or calcium alginates, propylene glycol alginate), gelatin, collagen and cellulose. Cellulose is a cellulose polymer that has been modified by reacting at least a portion of the hydroxyl groups on the sugar repeat units with a compound to form ester-linked or ether-linked substituents. For example, cellulose ethylcellulose has ether-linked ethyl groups attached to sugar repeat units And cellulose acetate has an ester-linked acetate substituent. In certain embodiments, cellulosic articles for use with an erodable matrix include water-soluble and water-erodable cellulosic articles, which may include, for example, Ethyl Cellulose (EC), Methyl Ethyl Cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), Cellulose Acetate (CA), Cellulose Propionate (CP), Cellulose Butyrate (CB), Cellulose Acetate Butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC). In certain embodiments, the cellulose comprises various grades of low viscosity (MW less than or equal to 50,000 daltons, e.g., american dow chemical methyl cellulose (dow methyl cellulose)^TM) Series E5, E15LV, E50LV and K100LY) and high viscosity (MW greater than 50,000 daltons, e.g., E4MCR, E10MCR, K4M, K15M and K100M and methylcellulose (methylcellulose)^TM) K series) HPMC. Other commercially available HPMC types include Shin Etsu hypromellose 90SH series.

Other materials that may be used as erodible matrix materials include, but are not limited to, pullulan, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid esters, polyacrylamides, polyacrylic acid, copolymers of ethylacrylic acid or methacrylic acid (R) ((R))

U.S. Rohm and Haas Inc. (Rohmamerica, Inc.), Piscataway, New Jersey, and other acrylic acid derivatives such as butyl methacrylate, methyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl) methacrylate, and (trimethylaminoethyl) methacrylate chloride homopolymers and copolymers.

Alternatively, the agents of the present invention may be applied via a non-aggressive matrix or incorporated into a non-aggressive matrix device for administration. In such devices, the reagents described herein are distributed in an inert matrix. The agent is released by diffusion through an inert matrix. Examples of materials suitable for the inert matrix include insoluble plastics (e.g., methyl acrylate-methyl methacrylate copolymer, polyvinyl chloride, polyethylene), hydrophilic polymers (e.g., ethyl cellulose, cellulose acetate, cross-linked polyvinylpyrrolidone (also known as crospovidone)), and aliphatic compounds (e.g., carnauba wax, microcrystalline wax, and triglycerides). Such devices are described in remington: pharmaceutical Science and Practice (Remington: The Science and Practice of Pharmacy), 20 th edition (2000).

As noted above, the reagents described herein may also be incorporated into an osmotic control device. Such devices typically include a core containing one or more agents as described herein and a water permeable, insoluble and non-aggressive coating surrounding the core that controls the flow of water from the aqueous environment of use into the core to cause drug release by way of some or all of the core being extruded into the environment of use. In certain embodiments, the coating is polymeric, water permeable, and has at least one delivery port. The core of the osmotic engine optionally includes an osmotic agent that imbibes water from the surrounding environment through the semipermeable membrane. The osmotic agent contained in the core of the device may be a water swellable hydrophilic polymer, or an osmogen (osmogen), also known as an osmotic agent (osmagent). Pressure is created within the device forcing the reagent out of the device via an orifice sized to minimize solute diffusion while preventing the build-up of an hydrostatic head. Non-limiting examples of osmotic control devices are disclosed in U.S. patent application serial No. 09/495,061.

The amount of water-swellable hydrophilic polymer present in the core may range from about 5 to 80 wt.% (including, for example, 10 to 50 wt.%). Non-limiting examples of core materials include hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly (2-hydroxyethyl methacrylate), poly (acrylic acid), poly (methacrylic acid), polyvinylpyrrolidone (PVP) and cross-linked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers and PVA/PVP copolymers with hydrophobic monomers, such as methyl methacrylate, vinyl acetate, etc., hydrophilic polyurethanes including long PEO blocks, croscarmellose sodium, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum and sodium starch glycolate. Other materials include hydrogels comprising interpenetrating networks of polymers that may be formed by addition or by polycondensation, the components of which may comprise hydrophilic and hydrophobic monomers, such as those just mentioned. Water swellable hydrophilic polymers include, but are not limited to, PEO, PEG, PVP, croscarmellose sodium, HPMC, sodium starch glycolate, polyacrylic acid, and crosslinked forms or mixtures thereof.

The core may also include an osmogen (or osmotic agent). The amount of osmogen present in the core may be about 2 to 70 wt.% (including, for example, 10 to 50 wt.%). Typical types of suitable osmotic agents are water-soluble organic acids, salts and sugars, which are capable of absorbing water, thereby creating an osmotic pressure gradient across the barrier layer of the surrounding coating. Typical useful osmogens include, but are not limited to, magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, sodium sulfate, mannitol, xylitol, urea, sorbitol, inositol, raffinose, sucrose, glucose, fructose, lactose, citric acid, succinic acid, tartaric acid, and mixtures thereof. In certain embodiments, the osmogen is glucose, lactose, sucrose, mannitol, xylitol, sodium chloride, including combinations thereof.

The rate of drug delivery is controlled by factors such as the permeability and thickness of the coating, the osmotic pressure of the drug-containing layer, the degree of hydrophilicity of the hydrogel layer, and the surface area of the device. One skilled in the art will appreciate that increasing the thickness of the coating will decrease the release rate, while any of the following will increase the release rate: increasing the permeability of the coating; increasing the hydrophilicity of the hydrogel layer; increasing the osmotic pressure of the drug-containing layer; or to increase the surface area of the device.

In certain embodiments, it is desirable that particles of the agents described herein be entrained in the extruded fluid during operation of such osmotic devices. In order to enable the particles to be sufficiently entrained, before the particles have had a chance to settle in the tablet coreThe dosage form is dispersed in the fluid. One way to achieve this is by adding a disintegrant that is used to break the compressed core into its granular components. Non-limiting examples of standard disintegrants include sodium starch glycolate (e.g., sodium starch)^TMCLV), microcrystalline cellulose (e.g. microcrystalline cellulose)^TM) Microcrystalline silicified cellulose (e.g. panthenol)^TM) And croscarmellose sodium (e.g., Ac-Di-Sol)^TM) And other disintegrants known to those skilled in the art. Depending on the particular formulation, some disintegrants are preferred over others. Some disintegrants can hinder drug delivery from the device by swelling with water to form a gel. The non-gelling, non-swelling disintegrant provides for more rapid dispersion of the drug particles within the core as water enters the core. In certain embodiments, the non-gelling, non-swelling disintegrant is a resin, such as an ion exchange resin. In one embodiment, the resin is ambolaite^TMIRP 88 (available from Rohm and Haas, Philadelphia, Pa.). When used, the disintegrant is present in an amount of about 1-25% of the core agent.

Another example of an osmotic device is an osmotic capsule. The capsule shell or a portion of the capsule shell may be semipermeable. The capsule may be filled with a powder or liquid consisting of the agent described herein, an excipient that absorbs water to provide an osmotic potential and/or a water swellable polymer, or an optional solubilizing excipient. The capsule core may also be made by: with bilayer or multilayer reagents similar to the bilayer, trilayer or concentric geometry described above.

Another type of osmotic device useful in the present invention includes coated expandable tablets such as described in EP 378404. Coated swellable tablets comprise a tablet core comprising an agent as described herein and a swelling material, preferably a hydrophilic polymer, coated with a membrane comprising holes, or pores, through which the hydrophilic polymer can extrude and carry a pharmaceutical agent in an aqueous use environment. Alternatively, the membrane may contain polymeric or low molecular weight water-soluble porogens. The porogen dissolves in the aqueous environment, creating pores through which the hydrophilic polymer and agent can pass and be extruded. Examples of porogens are water-soluble polymers such as HPMC, PEG and low molecular weight compounds such as glycerol, sucrose, glucose and sodium chloride. In addition, holes may also be formed in the coating by drilling holes in the coating using a laser or other mechanical method. In such osmotic devices, the membrane material may comprise any membrane-forming polymer, including water-permeable or impermeable polymers, provided that the membrane deposited on the core of the sheet is porous or contains water-soluble porogens or macroscopic pores with water intrusion and drug release. Embodiments of this type of sustained release device may also be multilayered, for example as described in EP 378404.

When the agent described herein is a liquid or oil, such as the lipid vehicle formulation described in WO05/011634, the osmotic controlled release device may comprise a soft gel or gelatin capsule formed with a composite wall and comprising a liquid formulation, wherein the wall comprises a barrier layer formed on an outer surface of the capsule, an expandable layer formed on the barrier layer, and a semipermeable layer formed on the expandable layer. The delivery port connects the liquid formulation with an aqueous use environment. Such devices are described in e.g. US6419952, US6342249, US5324280, US4672850, US4627850, US4203440 and US 3995631.

As further noted above, the agents described herein may be provided in particulate form, typically ranging in size from about 10 μm to 2mm (including, for example, from about 100 μm to 1mm in diameter). Such multiparticulates may be packaged, for example, in a capsule (such as a gelatin capsule or a capsule formed from a water soluble polymer such as HPMCAS, HPMC or starch); dosing as a suspension or slurry in a liquid; or they may be formed into tablets, caplets, or pills by compression or other methods known in the art. Such multiparticulates can be prepared by any known method, such as wet and dry granulation, extrusion/spheronization, roller compaction, melt solidification or coating the seed core by spray coating. For example, in wet and dry granulation processes, the agents described herein and optional excipients may be granulated to form multiparticulates of the desired size.

The reagents may be incorporated into microemulsions, which are typically thermodynamically stable, isotropic transparent dispersions of two immiscible liquids (e.g., oil and water) stabilized by an interfacial film of surfactant molecules (Encyclopedia of formulation Technology, New York (New York): Marcel Dekker, 1992, volume 9). For the preparation of microemulsions, surfactants (emulsifiers), cosurfactants (coemulsifiers), oil and water phases are required. Suitable surfactants include any surfactant that can be used to prepare an emulsion, such as the emulsifiers typically used to prepare creams. The co-surfactant (or "co-emulsifier") is typically selected from polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier/co-emulsifier combinations are not generally necessarily selected from the following group: glyceryl monostearate and polyoxyethylene stearate; polyethylene glycol and ethylene glycol palmitate stearate; and caprylic and capric triglycerides and oleoyl macrogolglycerides. The aqueous phase will typically include not only water but also buffers, glucose, propylene glycol, polyethylene glycol, preferably low molecular weight polyethylene glycols (e.g., PEG300 and PEG400) and/or glycerol and the like, while the oil phase will typically include, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono-, di-and triglycerides, mono-and di-esters of PEG (e.g., oleoyl polyglycolyglycerides) and the like.

The compounds described herein can be incorporated into pharmaceutically acceptable nanoparticle, nanosphere, and nanocapsule formulations (Delie and Blanco-Prieto, 2005, Molecule (Molecule) 10: 65-80). Nanocapsules can generally trap compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, ultrafine particles (about 0.1 μm in size) can be designed using polymers that can be degraded in vivo (e.g., biodegradable polyalkylcyanoacrylate nanoparticles). Such particles are described in the prior art.

Another embodiment of the invention is an implantable device coated with a compound of the invention. The compounds may also be coated onto implantable medical devices, such as beads, or co-formulated with polymers or other molecules to provide a "drug depot" that allows the drug to be released over a longer period of time than when an aqueous solution of the drug is administered. General preparation of suitable coatings and coated implantable devices are described in U.S. patent nos. 6,099,562; 5,886,026, respectively; and 5,304,121. The coating is typically a biocompatible polymeric material such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate and mixtures thereof. The coating may optionally be further covered with a suitable topcoat of fluorosilicone, polysaccharide, polyethylene glycol, phospholipid or combinations thereof to impart controlled release characteristics to the composition.

Formulations include those suitable for the routes of administration detailed herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are commonly found in Remington's. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers and/or finely divided solid carriers and then, if necessary, shaping the product.

The term "administration" or "administration" with respect to a compound, composition or formulation of the present invention refers to introducing the compound into an animal system in need of treatment. When a compound of the invention is provided in combination with one or more other active agents, "administering" and variations thereof are each understood to include the simultaneous and/or sequential introduction of the compound and the other active agent.

The compositions described herein may be administered systemically or locally, for example: oral (e.g., using capsules, powders, solutions, suspensions, tablets, sublingual tablets, etc.), by inhalation (e.g., using aerosols, gases, inhalers, nebulizers, etc.), otic (e.g., using ear drops), topical (e.g., using creams, gels, liniments, lotions, ointments, pastes, transdermal patches, etc.), ophthalmic (e.g., using eye drops, ophthalmic gels, ophthalmic ointments), rectal (e.g., using enemas or suppositories), nasal, buccal, vaginal (e.g., using lavages, intrauterine devices, pessaries or tablets, etc.), via implanted reservoirs, etc., or parenteral, other means depending on the severity and type of the condition being treated. The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally, or intravenously.

The pharmaceutical compositions described herein may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) dissolution retardants, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) adsorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. Tablets may be uncoated or they may be coated by known techniques including microencapsulation to mask unpleasant tastes or to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed. Water soluble taste masking materials such as hydroxypropyl methylcellulose or hydroxypropyl cellulose may be used.

Formulations of the compounds of table 1A or table 1B suitable for oral administration may be prepared as discrete units such as tablets, pills, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules such as gelatin capsules, syrups or elixirs. Formulations of the compounds for oral use may be prepared according to any method known in the art for the preparation of pharmaceutical compositions.

Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be prepared by compression molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin; or soft gelatin capsules wherein the active ingredient is mixed with a water-soluble carrier, such as polyethylene glycol, or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

The active compound may also be formed into microencapsulated forms with one or more excipients as described above.

When an aqueous suspension is desired for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents may be added. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring agent, a coloring agent and an antioxidant.

Sterile injectable forms of the compositions described herein (e.g., for parenteral administration) can be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable carriers and solvents that may be used are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as tweens, spans and other emulsifying agents or bioavailability enhancers which are commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms may also be used for injection formulation purposes.

Oily suspensions may be formulated by suspending the compounds of table 1A or table 1B in a vegetable oil (for example arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (for example liquid paraffin). The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as butylated hydroxyanisole or alpha-tocopherol.

Aqueous suspensions of the compounds in table 1A or table 1B contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, for example sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; and dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives (e.g. ethyl or propyl p-hydroxybenzoate), one or more colouring agents, one or more flavouring agents and one or more sweetening agents, such as sucrose or saccharin.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium for use.

To extend the efficacy of the compounds described herein, it is often desirable to slow the absorption of the compounds in subcutaneous or intramuscular injections. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of such compounds depends on their rate of dissolution, which in turn depends on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered compound form is achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot forms are prepared by forming microencapsulated matrices of the compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer used, the rate of release of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions which are compatible with body tissues.

The injectable solution or microemulsion may be injected into the bloodstream of a patient by a local bolus injection. OptionallyIt may be advantageous to apply the solution or microemulsion in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is Deltec CADD-PLUS^TMModel 5400 intravenous pump.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, beeswax, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Other formulations suitable for vaginal administration may be pessaries, tampons, creams, gels, pastes, foams or spray formulations.

The pharmaceutical compositions described herein may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including eye, ear, skin or lower intestinal tract diseases. It is readily possible to prepare suitable topical formulations for each of these regions or organs.

Dosage forms for topical or transdermal administration of the compounds described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. It may be desirable to combine the active ingredient under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers. Ophthalmic formulations, ear drops and eye drops are also within the scope of the invention. In addition, the present invention contemplates the use of transdermal patches, which have the advantage of providing controlled delivery of the compound to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. Topical application to the lower intestinal tract may be carried out in rectal suppository formulations (see above) or in suitable enema formulations. Topical transdermal patches may also be used.

For topical application, the pharmaceutical compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or preferably, as solutions in isotonic, pH adjusted sterile saline, with or without preservatives, such as benzylalkyl ammonium chloride. Alternatively, for ophthalmic use, the pharmaceutical compositions may be formulated as ointments, such as petrolatum. For the treatment of the eye or other external tissues, such as the mouth and skin, the formulations may be applied as a topical ointment or cream containing, for example, from 0.075 to 20% by weight of the active ingredient. When formulated in an ointment, the active ingredient may be used with an oil-based, paraffin-based or water-miscible ointment base.

Alternatively, the active ingredient may be formulated as a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include polyhydric alcohols, i.e., alcohols having two or more hydroxyl groups, such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerol and polyethylene glycols (including PEG 400) and mixtures thereof. Desirably, the topical formulation may include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin permeation enhancers include dimethyl sulfoxide and related analogs.

The oil phase of emulsions prepared using the compounds in table 1A or table 1B may be composed of known ingredients in a known manner. Although the phase may contain only emulsifiers (otherwise known as emulsifiers), it desirably contains at least one emulsifier and a fatFat, oil or a mixture of both fat and oil. The hydrophilic emulsifier may be included together with a lipophilic emulsifier as a stabilizer. In some embodiments, the emulsifier comprises an oil and a fat. At the same time, emulsifiers with or without stabilizers together constitute the so-called emulsifying wax, and the wax together with oils and fats constitutes the so-called emulsifying ointment base, from which the oily dispersed-phase formulation of a cream is formed. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the compounds in Table 1A or Table 1B include Tween^TM-60, span^TM-80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.

The pharmaceutical compositions may also be administered by nasal aerosol or by inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other conventional solubilizing or dispersing agents. Formulations suitable for intrapulmonary or intranasal administration have a particle size, for example in the range of 0.1 to 500 microns (including particles in the range between 0.1 and 500 microns in increments of microns, e.g. 0.5, 1, 30, 35 microns etc.), which is rapidly inhaled through the nasal passages or orally inhaled to achieve administration of the alveolar sacs.

Pharmaceutical compositions (or formulations) for use can be packaged in a variety of ways, depending on the method used to administer the drug. Typically, the article of manufacture for dispensing comprises a pharmaceutical formulation deposited in a suitable form in a container. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), pouches, ampoules, plastic bags, metal cylinders and the like. The container may also include tamper-proof means to prevent inadvertent touching of the contents of the package. In addition, the container is labeled with a label describing the contents of the container. The tag may also include an appropriate warning.

The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for immediate use for injection. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the type previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as described above, or an appropriate fraction thereof, of the active ingredient.

In another aspect, a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof, can be formulated in a veterinary composition comprising a veterinary carrier. Veterinary carriers are materials used for the purpose of administering the composition and can be solid, liquid or gaseous materials which are inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

Method of treatment

In another aspect, the present invention relates to the treatment of certain diseases by the use of sGC stimulators, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising them, alone or in combination, in a patient in need thereof.

The present disclosure relates to stimulators of soluble guanylate cyclase (sGC), pharmaceutical formulations thereof and their use, alone or in combination with one or more additional agents, for the treatment and/or prevention of various diseases, wherein an increase in the concentration of NO or the concentration of cGMP is desirable.

An increase in NO or cGMP concentration produced in the tissue can lead to vasodilation, thereby inhibiting platelet aggregation and adhesion, producing an antihypertensive effect, an anti-remodeling effect, an anti-fibrotic, an anti-apoptotic effect, an anti-inflammatory effect, a neuronal signal transmission effect, and the like.

In other embodiments, the compounds disclosed herein are sGC stimulators that are useful for the prevention and/or treatment of diseases and disorders characterized by bioavailability of NO and/or an undesirable reduction in NO sensitivity in a biological system (e.g., a human), such as those associated with oxidative or nitrosative stress.

The term "cardiovascular disease" (or "cardiovascular disorder") as used herein refers to a disease based on abnormal symptoms of the circulatory organs such as the heart, blood vessels (arteries, capillaries and veins) or heart and blood vessels. The term also includes any disease that commonly affects the cardiovascular system, including heart disease, cerebrovascular disease, vascular disease of the kidneys, liver and related organs, or pulmonary and peripheral arterial disease, among others.

An "sGC-associated cardiovascular disease" is a cardiovascular disease known or suspected to involve the NO/sGC/cGMP system and is a cardiovascular disease that can be treated or prevented by sGC activation/stimulation, by activating NO synthase, or by adding NO or NO-donors or NO precursors such as L-arginine or L-citrulline, or by inhibiting the PDE (phosphodiesterase) enzyme responsible for cGMP breakdown, or any combination of the above methods.

The term "vasodilation" as used herein refers to widening of blood vessels. It results from the relaxation of smooth muscle cells within the vessel wall, particularly in the large veins, large arteries and smaller arterioles. Essentially, this process is in contrast to "vasoconstriction", which is the narrowing of a blood vessel. When a blood vessel is dilated, the flow of blood increases due to the decrease in vascular resistance. Thus, the expansion of arterial blood vessels (primarily arterioles) reduces blood pressure. The response may be intrinsic (local processes in the surrounding tissue) or extrinsic (hormones or nervous system). Furthermore, the response may be localized to a particular organ (depending on the metabolic needs of a particular tissue, such as during strenuous exercise), or it may be systemic (cycling through the system).

The term "vasoconstriction" as used herein refers to the narrowing of blood vessels due to muscle contraction. Vasoconstriction is a mechanism by which the body regulates and maintains Mean Arterial Pressure (MAP). Systemic vasoconstriction usually results in an increase in systemic blood pressure, but it may also occur in specific tissues, resulting in a local decrease in blood flow.

As used herein, the term "bronchoconstriction" is used to define the constriction of the airways in the lungs, due to tightening of the surrounding smooth muscles, with consequent occurrence of coughing, wheezing and shortness of breath. There are a number of causes for this condition, the most common being asthma. Movement and allergies may lead to other disorders in asymptomatic individuals. Other disorders such as Chronic Obstructive Pulmonary Disease (COPD) may also be accompanied by bronchoconstriction.

Herein, the terms "hypertension", "arterial hypertension" or "Hypertension (HBP)" are used interchangeably and refer to an extremely common and highly preventable chronic condition in which the blood pressure in an artery is above normal or desirable. It represents an important risk factor for several serious cardiovascular and renal diseases if not properly controlled. Hypertension may be a primary disease referred to as "essential hypertension" or "essential hypertension", or it may be caused by or associated with other diseases, in which case it is classified as "secondary hypertension". Essential hypertension accounts for 90-95% of all cases.

The term "recalcitrant hypertension" as used herein refers to hypertension that remains above the target blood pressure (typically less than 140/90mmHg, although the recommended target is less than 130/80mmHg for patients with simultaneous diabetes or kidney disease) even if three antihypertensive drugs belonging to different classes of antihypertensive drugs are used simultaneously. People who need four or more drugs to control their blood pressure are also considered to have persistent hypertension. Hypertension is a very common comorbid condition in diabetes, affecting about 20-60% of diabetic patients depending on obesity, race and age. This type of hypertension is referred to herein as "diabetic hypertension". In type 2 diabetes, insulin resistance metabolic syndrome (also including central obesity and dyslipidemia) is present as part of the disease. In type 1 diabetes, hypertension may reflect the onset of diabetic nephropathy.

As used herein, "Pulmonary Hypertension (PH)" is a disease characterized by a sustained elevation of blood pressure in the pulmonary vessels (pulmonary arteries, pulmonary veins, and pulmonary capillaries), which can lead to right heart hypertrophy, ultimately leading to right heart failure and death. Common symptoms of PH include shortness of breath, dizziness and fainting, all of which are exacerbated by exertion. Without treatment, the average life expectancy after diagnosis was 2.8 years. There are many different forms of PH, which are classified according to their etiology, and the categories include Pulmonary Arterial Hypertension (PAH), PH with left heart disease, PH associated with pulmonary disease and/or hypoxemia, PH due to chronic thrombotic and/or embolic disease, and miscellaneous PH. PAH is rare in the general population, but the prevalence increases in association with certain common conditions such as HIV infection, scleroderma and sickle cell disease. Other forms of PH are generally more common than PAH, and for example, the association of PH with Chronic Obstructive Pulmonary Disease (COPD) is of particular interest. Current treatments for pulmonary hypertension depend on the stage and mechanism of the disease.

The term "coronary artery disease" refers to a condition in which the blood supply to the myocardium is partially or completely blocked (ischemia of the myocardium or myocardium). This reduction in blood supply to the myocardium may lead to many "acute myocardial syndromes": chest pain ("angina", stable or unstable) and different types of heart attacks ("myocardial infarction" or MI). One common cause of coronary artery disease is "atherosclerosis," which refers to arteriosclerosis due to fatty deposits in the arterial wall, then progresses through the formation of atherosclerotic plaques, narrowing and eventually blocking blood flow in the artery. This atherosclerotic process may also affect other arteries, not just the arteries of the heart. Blood clots are the most common cause of arterial obstruction, usually an artery that has been partially occluded by an atherosclerotic plaque (atheroma) that may rupture or tear, eventually leading to clot formation. Occasionally, coronary artery disease is caused by spasm of the coronary arteries, which may occur spontaneously or as a result of the use of certain drugs (e.g., cocaine, nicotine). Rarely, the causes of coronary artery disease are birth defects, viral infections (e.g., kawasaki disease), systemic lupus erythematosus (lupus erythematosus), arterial inflammation (arteritis), blood clots that migrate from the ventricles to the coronary arteries or blood clots that result from physical injury (e.g., from injury or radiation therapy).

As used herein, "unstable angina" refers to changes in the symptoms pattern of angina pectoris, including chronic or worsening angina and new episodes of severe symptoms.

MI can be divided into two types: "non-ST elevation" MI and "ST elevation" MI. The complications of acute coronary syndrome depend on the degree, time and location of coronary occlusion. If the obstruction affects a large number of heart muscles, the heart will not pump efficiently. If the obstruction blocks the electrical system of blood flow to the heart, the heart rhythm may be affected. When a heart attack occurs, a portion of the myocardium dies. Dead tissue and scar tissue replacing it do not contract. Scar tissue sometimes even dilates or bulges when the rest of the heart attempts to contract. Thus, there is less muscle to pump blood. If enough muscles die, the pumping capacity of the heart may be reduced, such that the heart fails to meet the body's oxygen and blood needs, heart failure, hypotension, or both may occur. If more than half of the myocardium is damaged or dies, the heart often fails to function, resulting in severe loss of function or death.

As used herein, "heart failure" (HF) is a progressive disorder of Left Ventricular (LV) myocardial remodeling, ultimately leading to a complex clinical syndrome in which impaired cardiac function and circulating congestion are well-characterized and result in inadequate delivery of blood and nutrients to body tissues. This occurs when the heart is damaged or over-working and is unable to pump all of the returning blood from the systemic circulation. As less blood is drawn, the blood returning to the heart flows backwards and body fluids accumulate in other parts of the body. Heart failure also impairs the ability of the kidney to process sodium and water, further complicating fluid retention. Heart failure is characterized by autonomic dysfunction, neurohormonal activation, and overproduction of cytokines, leading to progressive circulatory failure. Symptoms of heart failure include: dyspnea (tachypnea) at night when exercising or resting and waking up due to sudden dyspnea, both manifested as pulmonary edema; general fatigue or weakness; foot, ankle and leg edema; rapidly increasing weight; chronic cough, including the production of mucus or blood. According to its clinical manifestations, heart failure is classified as new onset, transient, acute, late acute or chronic. Acute heart failure (i.e., a rapid or gradual onset of symptoms requiring urgent treatment) can occur as a new onset or as a result of conversion to decompensated chronic heart failure. The term "heart failure" is commonly used to denote "chronic heart failure". The terms "Congestive Heart Failure (CHF)" or "congestive heart failure (CCF)" are often used interchangeably with chronic heart failure. Common causes of heart failure include coronary artery disease, including prior myocardial infarction (heart attack), hypertension, atrial fibrillation, valvular heart disease, and cardiomyopathy. These are heart failures caused by structural or functional changes in the heart.

There are two main types of heart failure: "heart failure due to reduced ejection fraction (HFREF)", also known as "heart failure due to left ventricular systolic dysfunction" or "systolic heart failure"; and "heart failure with preserved ejection fraction (HFPEF"), also known as "diastolic heart failure" or "heart failure with normal ejection fraction (HFNEF)". Ejection fraction is the proportion of blood in the heart that is pumped out of the heart during a single contraction. It is a percentage between 50% and 75% of the normal value.

The term "acute" (as in "acute HF") is used to denote a rapid onset, while "chronic" refers to a long duration. Chronic heart failure is a long-term condition, usually with stable therapeutic symptoms. "acute decompensated" heart failure is worsening or decompensated heart failure, meaning that a person can be characterized as having episodes that result in changes in the signs and symptoms of heart failure that require urgent treatment or hospitalization. Heart failure can also occur in the case of high output (which is referred to as "high output heart failure"), in which ventricular contraction function is normal, but the heart is unable to handle the important case of increased blood volume.

In cardiovascular physiology, the term "Ejection Fraction (EF)" is defined as the fraction of blood in the left and right ventricles pumped out in each heartbeat or cardiac cycle. In limited mathematics allowed for medical imaging, EF is applied to the right ventricle, which injects blood into the pulmonary circulation through the pulmonary valve; or the left ventricle, which ejects blood through the aortic valve into the brain and systemic circulation.

The term "heart failure with preserved ejection fraction (HFPEF)" is generally understood to refer to the manifestation of signs and symptoms of heart failure with an ejection fraction greater than 55%. It is characterized by a decrease in left ventricular compliance, resulting in an increase in pressure in the left ventricle. HFPEF often increases left atrial size due to left ventricular dysfunction. For congestive heart failure, the risk of atrial fibrillation and pulmonary hypertension increases. Risk factors are hypertension, hyperlipidemia, diabetes, smoking and obstructive sleep apnea. In this type of heart failure, the heart muscle contracts well, but the ventricles are not filled with blood during diastole.

The term "heart failure with reduced ejection fraction (HFREF)" refers to heart failure in which the ejection fraction is less than 40%.

Diabetes is a common complication in patients with heart failure and is associated with a poor prognosis and potentially affects the efficacy of treatment. Other important complications include systemic hypertension, chronic airflow obstruction, sleep apnea, cognitive dysfunction, anemia, chronic kidney disease and arthritis. Chronic left heart failure is often associated with the development of pulmonary hypertension. The frequency of certain complications varies by gender: in women, hypertension and thyroid disease are more common, while men are more commonly suffering from Chronic Obstructive Pulmonary Disease (COPD), peripheral vascular disease, coronary artery disease and renal insufficiency. Depression is a common complication of heart failure, and both conditions can and often complicate one another. Cachexia has long been recognized as a serious and frequent complication of heart failure, affecting up to 15% of heart failure patients and associated with poor prognosis. Cardiac cachexia is defined as a non-edematous, involuntary loss of body weight of at least 6% over a period of 6 months.

The term "arrhythmia" as used herein refers to an abnormal heart rhythm that occurs in more than 90% of people who have had a heart attack. Sometimes, the problem is that the heart triggers that part of the heart beat, the heart rate may be too slow, or the heart beat too fast or irregular. Sometimes, the pulse signal is not conducted from one part of the heart to another, and the heartbeat may slow down or stop. Furthermore, areas of the myocardium that are not dead but have poor blood flow may be vulnerable to activation. This leads to heart rhythm problems such as ventricular tachycardia or ventricular fibrillation. This may lead to cardiac arrest if the heart completely stops pumping.

"pericardium" is a bag or membrane that surrounds the heart. "pericarditis" or inflammation of the membrane may progress to a heart attack and may lead to fever, pericardial effusion, inflammation of the membranes covering the lungs (pleura), pleural effusion and joint pain. Other complications after a heart attack may include mitral valve failure, rupture of heart muscle, bulging on the wall of the ventricle (ventricular tumour), blood clots and hypotension.

The term "cardiomyopathy" refers to a gradual impairment of the structure and function of the muscle wall of a heart chamber. The main types of cardiomyopathy are dilation, hypertrophy and restriction. Cardiomyopathies often cause symptoms of heart failure, which may also cause chest pain, fainting and sudden death.

The terms "mitral regurgitation," "mitral insufficiency," or "mitral insufficiency" refer to the condition in which the mitral valve of the heart fails to close tightly, allowing blood to regurgitate within the heart. As a result, blood cannot flow effectively through the heart or the rest of the body, resulting in fatigue or shortness of breath.

The term "sleep apnea" refers to the most common sleep disordered breathing disorder. The condition is characterized by intermittent, periodic reduction or complete cessation of airflow, which may or may not involve obstruction of the upper airway. There are three types of sleep apnea: obstructive sleep apnea (the most common form), central sleep apnea and mixed sleep apnea.

"Central Sleep Apnea (CSA)" is caused by failure of the respiratory normal signal in the brain, rather than physical obstruction of the airway. The lack of respiratory action results in an increase in carbon dioxide in the blood, which may cause the patient to become hot. CSA is rare in the general population, but is relatively common in patients with systolic heart failure.

The terms "metabolic syndrome", "insulin resistance syndrome" or "syndrome X" as used herein refer to a group or population of metabolic disorders (abdominal obesity, elevated fasting glucose, "dyslipidemia", (i.e. elevated lipid levels) and elevated blood pressure (HBP)), which occur together more commonly than occasionally alone and which promote the development of type 2 diabetes and cardiovascular disease simultaneously. The metabolic syndrome is characterized by a specific lipid profile of increased triglycerides, decreased high density lipoprotein cholesterol (HDL-cholesterol) and in some cases moderately elevated low density lipoprotein cholesterol (LDL-cholesterol) levels, and accelerated development of "atherosclerotic disease" due to stress constituting part of the risk factors. There are several types of dyslipidemia: "hypercholesterolemia" refers to elevated cholesterol levels; familial hypercholesterolemia is a particular form of hypercholesterolemia caused by defects on chromosome 19(19p 13.1-13.3). "hypertriglyceridemia" refers to elevated levels of glycerides (e.g., "hypertriglyceridemia" refers to elevated triglyceride levels); "hyperlipoproteinemia" refers to elevated lipoprotein (usually LDL, unless otherwise indicated) levels.

The term "steatosis" refers to an abnormal retention of intracellular lipids. It often reflects impairment of the normal processes of synthesis and elimination of triglycerides. Excess fat accumulates in vesicles that displace the cytoplasm of the cell. In severe cases, cells may burst. Since the liver is an organ mainly associated with fat metabolism, steatosis is generally observed in the liver. It is also observed in heart, kidney and muscle tissue.

The term "Peripheral Vascular Disease (PVD)", also commonly referred to as "Peripheral Arterial Disease (PAD)" or "Peripheral Arterial Occlusive Disease (PAOD)" as used herein, refers to the occlusion of a coronary artery, the aortic arch vasculature or the aorta outside the brain. PVD can be caused by atherosclerotic, inflammatory processes leading to stenosis, embolism, thrombosis, or other types of occlusion. It leads to acute or chronic "ischemia (ischemia supply)". In general, PVD is a term used to refer to atherosclerotic obstructions found in the lower limb. PVD also includes a class of diseases classified as microvascular diseases caused by paroxysmal stenosis of an artery (e.g., "raynaud's phenomenon") or its dilatation (erythromelalgia), i.e., vasospasm. Peripheral arterial disease includes occlusive thromboangiitis, peripheral arterial occlusive disease, Raynaud's disease and Raynaud's syndrome. Common symptoms are cold legs or feet, intermittent claudication, lower limb pain and severe limb ischemia (lower limb ulcers and necrosis). Guidelines for the diagnosis and treatment of peripheral artery disease can be found in the european journal of vascular and vascular surgery (eur.j.vasco endovasc.surg), 2007, 33(1), S1.

The term "stenosis" as used herein refers to an abnormal narrowing in a blood vessel or other tubular organ or structure. It is also sometimes referred to as a "stenosis" (as in a urethral stricture). The term "narrowing" is synonymous, but is typically used only in the context of aortic narrowing. The term "restenosis" refers to the recurrence of stenosis after surgery.

The term "thrombosis" refers to the formation of blood clots ("thrombi") within a blood vessel that impede the flow of blood through the circulatory system. When a blood vessel is damaged, the body uses platelets (thrombocytes) and fibrin to form a blood clot to prevent blood loss. Alternatively, even if the blood vessel is not injured, if proper conditions exist, blood clots may form in the body. If the coagulation is too severe and the clot breaks, the moving clot is called an "embolism". The term "thromboembolism" refers to the combination of thrombosis and its major complication "embolism". When thrombus occupies more than 75% of the arterial lumen surface area, the blood flow provided to the tissue is reduced enough to cause symptoms due to a reduction in oxygen (hypoxia) and accumulation of metabolites such as lactic acid ("gout"). Over 90% of the blockages can lead to hypoxia, complete oxygen depletion and "infarction", a mode of cell death.

An "embolism" (multiple embolisms) is an event in which an embolus (a detached intravascular substance capable of occluding the arterial capillary bed at a location remote from its origin) enters the narrow capillaries of the arterial bed, which results in a distal occlusion of the body (vessel occlusion). This cannot be confused with a thrombus that is blocked at the primary site. The material forming the plug may have many different sources: if the material is blood, an "embolism" is referred to as a "thrombus"; the solid material may also include fat, bacterial debris, infected tissue, and the like.

"ischemia" is a limitation of the blood supply to a tissue, resulting in a deficiency of oxygen and glucose required for cellular metabolism (to keep the tissue viable). Ischemia is often caused by problems with blood vessels, resulting in tissue damage or tissue dysfunction. It also means ischemia in a particular part of the body, sometimes caused by congestion (e.g. vasoconstriction, thrombosis or embolism). If "ischemia" occurs in the myocardium (or "myocardium"), the ischemia is referred to as myocardial ischemia. Other types of ischemia include, for example, cerebral ischemia, critical limb ischemia, and the like.

"reperfusion" occurs when blood supply returns to the tissue after a period of ischemia. Upon return to the circulation of the tissue, inflammatory and oxidative stress processes may develop. An example of such a chain of events is ischemia reperfusion associated with organ transplantation.

"reperfusion injury" is tissue damage caused when blood supply returns to tissue after ischemia and inflammation, and subsequent oxidative damage occurs rather than restoration of normal function. The problem of ischemic reperfusion is often associated with microvascular damage, particularly due to increased permeability of capillaries and arterioles, resulting in increased diffusion across tissue and increased fluid filtration. Activated endothelial cells produce more reactive oxygen species, less NO after reperfusion, and this imbalance leads to an inflammatory response. Leukocytes carried to this area by the newly returned blood stream release many inflammatory factors and free radicals in response to tissue damage. The restored blood flow carries oxygen that damages cellular proteins, DNA and plasma membranes. This process of ischemia reperfusion is also thought to be responsible for the slowness and failure of wound healing (e.g., pressure sores or diabetic ulcers).

The term "vascular disease" as used herein is a general term for vascular (arterial, venous and capillary) diseases. The most common and prevalent vascular disease is "diabetic vascular disease," a common complication of chronic diabetes. Another common vascular disease is "cerebral amyloid angiopathy" (CAA), also known as neuroangiopathy, in which amyloid deposits form in the vessel walls of the central nervous system. The term congo red is used because the presence of abnormal aggregates of amyloid can be demonstrated by microscopic examination of brain tissue following the application of a special stain called congo red. Amyloid is only present in the brain, and thus the disease is not associated with other forms of amyloidosis.

A "stroke" or cerebrovascular accident (CVA) is a rapid loss of brain function due to interference with the blood supply to the brain. This may be due to "ischemia" (a lack of blood flow, thereby resulting in an inadequate supply of oxygen and glucose in the tissue) caused by occlusion (thrombosis, arterial embolism, fat accumulation or spasm) or hemorrhage (blood leakage). As a result, the affected area of the brain fails to function, which may result in one side of the body, one or more limbs failing to move, failing to understand or form speech, or failing to see one side of the field of view. Risk factors for stroke include increased age, hypertension, previous stroke or Transient Ischemic Attack (TIA), diabetes, high cholesterol, smoking and atrial fibrillation. Hypertension is the most important controllable risk factor for stroke. "ischemic stroke" is sometimes treated in hospitals by thrombolysis (also known as "clot lysis agent"), and some hemorrhagic strokes benefit from neurosurgery. Preventing relapse may involve administration of antiplatelet drugs (e.g., aspirin and dipyridamole) and the use of statins to control and reduce hypertension. Some patients may benefit from carotid endarterectomy and the use of anticoagulants.

"vascular dementia" is the second most common cause of dementia in the elderly. It is more common in men, usually beginning after age 70. More common in people with vascular risk factors (e.g. hypertension, diabetes, hyperlipidemia, smoking) and several strokes. Many people suffer from both vascular dementia and alzheimer's disease. Vascular dementia commonly occurs when multiple cerebellar infarcts (or sometimes hemorrhages) cause sufficient neuronal or axonal loss to impair brain function. Vascular dementia includes the following types: lacunar infarction (in which small blood vessels are affected and infarction occurs deep in the white and grey matter of the hemisphere); multi-infarct dementia (in which medium blood vessels are affected); strategic mono-infarct dementia (where the single infarct occurs in a critical area of the brain, such as the horn or thalamus; bingswanger dementia or subcortical arteriosclerotic encephalopathy (where small vascular dementia is associated with severe poorly controlled hypertension and systemic vascular disease, and which results in diffuse and irregular loss of axons and myelin, with extensive gliosis, tissue death due to infarction, or loss of blood supply to the white matter of the brain).

The term "glioma" refers to a type of tumor that begins in the brain or spinal cord. It is called glioma because it originates from glial cells. The most common site of glioma is the brain. Gliomas constitute about 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors.

According to the Diagnostic and Statistical Manual of Psychiatric Disorders of the American Psychiatric society, Fourth Edition (American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) (DSM-IV), the term "sexual dysfunction" includes a series of Disorders characterized by psychophysiological changes in sexual desire and imbalance associated with the sexual response cycle; while this type of problem is common, sexual dysfunction is considered to exist when the problem causes pain to the patient. Sexual dysfunction can be a physical or psychological cause. It may exist as a major disorder, usually hormonal in nature, although most commonly secondary to other medical disorders or for drug treatment of said disorders. All types of sexual dysfunction can be further classified as life-long, acquired, contextual or systemic (or combinations thereof).

DSM-IV-TR refers to five main categories of "female sexual dysfunction": disorders of libido/interest; "sexual arousal disorder (including genital, subjective and complex)"; orgasmic disorder; dyspareunia and vaginitis; and persistent sexual arousal disorder.

Female Sexual Arousal Disorder (FSAD) is defined as persistent or repetitive failure to obtain or maintain a sufficient level of sexual arousal, resulting in personal distress. FSAD includes lack of subjective sensory stimulation (i.e., subjective sexual arousal disorder) and lack of somatic responses such as lubrication and swelling (i.e., genital/physical sexual arousal disorder). FSAD can be a strict psychological cause, although it is usually caused or complicated by medical or physiological factors. Estrogen deprivation is the most common physiological condition associated with FSAD, which leads to urogenital atrophy and a reduction in vaginal lubrication.

As used herein, "Erectile Dysfunction (ED)" is male sexual dysfunction characterized by the inability to develop or maintain a penile erection during sexual performance. Penile erection is the hydraulic action of blood entering and remaining in the cavernous body within the penis. This process is usually initiated by sexual arousal when signals are transmitted from the brain to the nerves in the penis. When an erection is difficult to produce, erectile dysfunction is indicated. The most important organic causes are cardiovascular disease and diabetes, neurological problems (e.g., trauma from prostatectomy surgery), hormone deficiency (hypogonadism) and drug side effects.

In one embodiment, the compounds of table 1A or table 1B and pharmaceutically acceptable salts thereof as sGC stimulators are therefore useful for the prevention and/or treatment of the following types of cardiac, pulmonary, peripheral, hepatic, renal or cerebrovascular/endothelial disorders, conditions and diseases associated with the circulation:

diseases associated with hypertension and reduced coronary blood flow; acute and chronic coronary blood pressure elevation; arterial hypertension; vascular disease caused by cardiac and renal complications; vascular disease caused by heart disease, stroke, cerebral ischemia or renal failure; drug-resistant hypertension; diabetic hypertension; primary hypertension; secondary hypertension; gestational hypertension; pre-eclampsia; portal hypertension; myocardial infarction;

heart failure, HFPEF, HFREF; acute and chronic heart failure; more specific forms of HF: acute decompensated HF, right ventricular failure, left ventricular failure, total HF, ischemic cardiomyopathy, dilated cardiomyopathy, congenital heart defect, valvular defect HF, mitral stenosis, mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary insufficiency, valvular merger defect; diabetic heart failure; alcoholic cardiomyopathy or storage cardiomyopathy; dilatant HF, contractive HF; acute phase of existing chronic HF (worsening HF); diastolic or systolic dysfunction; coronary insufficiency; cardiac arrhythmia; reducing ventricular load; cardiac hypertrophy; heart failure/myocardial syndrome; portal hypertension; endothelial dysfunction or damage; atrioventricular rhythm and conduction disorders: class I-III atrioventricular block (AVBI-III), supraventricular tachycardia acceleration, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, ventricular tachycardia, torsade de pointes, atrial and ventricular premature beats, AV node extrasystoles, sick sinus syndrome, syncope, AV node refolding tachycardia; Wolff-Parkinson-White (Wolff-parkinsonon-White) syndrome or acute coronary syndrome; boxer cardiomyopathy; early ventricular contraction; cardiomyopathy; cancer-induced cardiomyopathy;

Thromboembolic disorders and ischemia; myocardial ischemia; infarction; myocardial infarction; heart disease; cardiac insufficiency; endothelial dysfunction; stroke; transient Ischemic Attack (TIA); obstructive thrombotic vasculitis; stable or unstable angina; coronary or peripheral arterial spasm; variant angina pectoris; prinzmetal (Prinzmetal) type angina; cardiac hypertrophy; pre-eclampsia; thrombotic disorders; ischemia reperfusion injury; ischemia reperfusion associated with organ transplantation; ischemia reperfusion related to lung transplantation, heart transplantation, graft failure; preserving blood substituents in trauma patients;

peripheral vascular disease; peripheral arterial disease; peripheral occlusive arterial disease; hypermyotonia; raynaud's syndrome or phenomenon (primary and secondary); raynaud's disease; critical limb ischemia; a peripheral plug; intermittent claudication; vaso-occlusive crisis; muscular dystrophy, duchenne muscular dystrophy, becker muscular dystrophy; a microcirculation abnormality; control of vascular leakage or permeability; lumbar spinal stenosis; occlusive thromboangiitis; thromboangiitis; peripheral perfusion disorder; arterial and venous thrombosis; microalbuminuria; peripheral and autonomic neuropathy; diabetic microangiopathy;

Edema; renal edema due to heart failure;

alzheimer's disease; parkinson's disease; vascular dementia; vascular cognitive disorders; cerebral vasospasm; congenital myasthenia syndrome; subarachnoid hemorrhage; traumatic brain injury; cognitive disorders such as those occurring in mild cognitive impairment, age-related learning and memory disorders, age-related memory loss, vascular dementia, head injury, stroke, post-stroke dementia, post-traumatic head injury, cognitive, concentration, learning or memory improvement following general and concentration disorders in children with learning and memory problems; dementia with lewy bodies; frontal dementia including pick's syndrome; progressive nuclear palsy; dementia with corticobasal degeneration; amyotrophic Lateral Sclerosis (ALS); huntington's disease; demyelination; multiple sclerosis; thalamic degeneration; Creutzfeldt-Jakob dementia; HIV dementia; schizophrenia accompanied by dementia or Korsakoff (Korsakoff) psychosis; multisystemic atrophy and other forms of parkinsonism; movement disorders; neuroprotection; anxiety, stress and depression or Post Traumatic Stress Disorder (PTSD); bipolar disorder; schizophrenia; CNS-related dysfunction and sleep disorders; pathological eating disorders and the use of luxury foods and addictive drugs; controlling cerebral perfusion; migraine headache; prevention and control of the consequences of cerebral infarction (brain relapse); preventing and controlling the consequences of stroke, cerebral ischemia and head injury;

Shock; cardiogenic shock; sepsis; septic shock; anaphylactic shock; an aneurysm; controlling leukocyte activation; inhibiting or modulating platelet aggregation; multiple Organ Dysfunction Syndrome (MODS); multiple Organ Failure (MOF);

lung/respiratory tract diseases: pulmonary Hypertension (PH); pulmonary Arterial Hypertension (PAH) and associated pulmonary vascular remodeling; vascular remodeling in the form of local thrombosis and right heart hypertrophy; hypertension of lung muscles; primary pulmonary hypertension; secondary pulmonary hypertension; familial pulmonary hypertension; sporadic pulmonary hypertension; anterior capillary pulmonary hypertension; idiopathic pulmonary hypertension; other forms of PH; PH associated with left ventricular disease, HIV, SCD, thromboembolism (CTEPH), sarcoidosis, COPD, pulmonary fibrosis, Acute Respiratory Distress Syndrome (ARDS), acute lung injury, alpha-1-antitrypsin deficiency (AATD), emphysema, smoking-induced emphysema and Cystic Fibrosis (CF); thrombotic pulmonary artery disease; a cluster pulmonary artery disease; cystic fibrosis; bronchoconstriction or pulmonary bronchoconstriction; acute respiratory distress syndrome; pulmonary fibrosis, lung transplantation; asthma.

Pulmonary hypertension associated with or related to: left ventricular dysfunction, hypoxemia, hypertension of type I, II, III, IV and V in world health organization, mitral valve disease, stenotic pericarditis, aortic valve stenosis, cardiomyopathy, mediastinal fibrosis, pulmonary venous return abnormality, pulmonary venous occlusive disease, pulmonary vasculitis, collagen vascular disease, congenital heart disease, pulmonary venous hypertension, interstitial lung disease, sleep disordered breathing, sleep apnea, alveolar hypoventilation, long-term exposure to high altitude, neonatal lung disease, capillary dysplasia, sickle cell disease, other blood clotting disorders, chronic thromboembolism, pulmonary embolism; pulmonary embolism due to tumor, parasite or foreign matter; sarcoidosis, lupus nephritis, schistosomiasis, sarcoidosis, chronic obstructive pulmonary disease, asthma, emphysema, chronic bronchitis, pulmonary capillary angiomatosis, histiocytosis X, lymphangiomatosis, compressed pulmonary vessels; compressed pulmonary blood vessels due to adenopathy, tumors or fibrosing mediastinitis;

Arteriosclerotic diseases or disorders: atherosclerosis; atherosclerosis associated with endothelial injury, platelet and monocyte adhesion and aggregation, smooth muscle proliferation or migration; restenosis; restenosis that occurs following thrombolytic therapy, Percutaneous Transluminal Angioplasty (PTA), transluminal coronary angioplasty (PTCA), heart transplantation, bypass surgery or inflammatory procedures;

microvascular and macrovascular lesions (vasculitis); fibrinogen levels and low density DLD increase; elevated plasminogen activator inhibitor 1(PA-1) concentration;

metabolic syndrome; metabolic diseases or diseases associated with metabolic syndrome: obesity; excess subcutaneous fat; (ii) excessive obesity; diabetes mellitus; hypertension; lipid-related disorders, hyperlipidemia, dyslipidemia, hypercholesterolemia, low high density lipoprotein cholesterol (HDL-cholesterol), moderately elevated low density lipoprotein cholesterol (LDL-cholesterol) levels, hypertriglyceridemia, hypolipoproteinemia, sitosterolemia, fatty liver disease, hepatitis; pre-eclampsia; polycystic kidney disease progression; hepatic steatosis or abnormal lipid accumulation in the liver; steatosis in the heart, kidney or muscle; alphabets proteinemia; sitosterolemia; xanthomatosis; tangjier's disease; hyperammonemia and related diseases; hepatic encephalopathy; other toxic encephalopathies; right (Reye) syndrome;

Sexual, gynecological and urological disorders: erectile dysfunction; impotence; premature ejaculation; female sexual dysfunction; female sexual arousal dysfunction; active sexual arousal disorder; vaginal atrophy; the sexual feeling is not quick; atrophic vaginitis; benign Prostatic Hyperplasia (BPH), prostatic hypertrophy, prostatic hyperplasia; bladder outlet obstruction; bladder Pain Syndrome (BPS); interstitial Cystitis (IC); overactive bladder; neurogenic bladder and incontinence; diabetic nephropathy; primary and secondary dysmenorrhea; lower Urinary Tract Syndrome (LUTS); endometriosis; pelvic pain; benign and malignant diseases of the male and female genitourinary organs;

chronic kidney disease; acute and chronic renal insufficiency; acute and chronic renal failure; lupus nephritis; potential or related renal diseases: hypoperfusion, intracellular hypotension, obstructive neuropathy, glomerulopathy, glomerulonephritis, acute glomerulonephritis, glomerulosclerosis, tubulointerstitial disease, renal disease, primary and congenital renal disease, nephritis; diseases characterized by abnormally reduced creatinine and/or water excretion; diseases characterized by abnormally elevated blood levels of urea, nitrogen, potassium and/or creatinine; diseases characterized by an alteration in renal enzyme activity, diseases characterized by an alteration in glutamyl synthase activity; diseases characterized by changes in urine osmolality or urine volume; diseases characterized by increased microalbuminuria, diseases characterized by albuminuria; diseases characterized by glomerular and arteriolar injury, tubular dilation, hyperphosphatemia, and/or the need for dialysis; sequelae of renal insufficiency; lung perfusion associated with renal insufficiency; renal insufficiency associated with HF; renal insufficiency associated with uremia or anemia; electrolyte disorders (hyperkalemia, hyponatremia); disorders of bone and carbohydrate metabolism;

Ocular diseases or disorders, such as glaucoma, retinopathy and diabetic retinopathy.

The term "inflammation" refers to the complex biological response of vascular tissue to harmful stimuli such as pathogens, damaged cells, or irritants. Typical signs of acute inflammation are pain, heat, redness, swelling and loss of function. Inflammation is a protective attempt by organisms to remove harmful stimuli and initiate the healing process. Inflammation is not a synonym for infection, even if both are often related (the former is usually the result of the latter). Inflammation can also occur in the absence of infection, although this type of inflammation is often maladaptive (e.g., in atherosclerosis). Inflammation is a patterned response and is therefore considered a mechanism of innate immunity, as compared to adaptive immunity specific for each pathogen. Without inflammation, the progressive destruction of tissue can impair the survival of the organism. On the other hand, chronic inflammation may lead to a number of diseases, such as pollinosis, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer (e.g. gallbladder cancer). It is for this reason that inflammation is often tightly regulated by the body. Inflammation can be classified as acute or chronic. "acute inflammation" is the initial response of the body to a noxious stimulus and is achieved by an increase in the movement of plasma and leukocytes (especially granulocytes) from the blood into the damaged tissue. The cascade of biochemical events spreads and matures the inflammatory response, involving the local vasculature, the immune system and various cells within the damaged tissue. Chronic inflammation, known as "chronic inflammation", results in a progressive change in the cell types present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissues from the inflammatory process.

In another embodiment, the compounds of table 1A or table 1B and pharmaceutically acceptable salts thereof as sGC stimulators are therefore useful for the prevention and/or treatment of the following types of cardiac, pulmonary, peripheral, hepatic, renal, digestive or central nervous system disorders, conditions and diseases that may be involved in inflammation or inflammatory processes:

myocardial inflammation (myocarditis); chronic myocarditis; acute myocarditis; viral myocarditis;

vasculitis; pancreatitis; peritonitis; rheumatoid diseases;

inflammatory diseases of the kidney; immune renal disease: kidney transplant rejection, immune complex-induced kidney disease, toxin-induced kidney disease, contrast-mediated kidney disease; diabetic and non-diabetic nephropathy, pyelonephritis, renal cyst, nephrosclerosis, hypertensive nephrosclerosis and nephrotic syndrome;

chronic interstitial inflammation, Inflammatory Bowel Disease (IBD), crohn's disease, Ulcerative Colitis (UC);

inflammatory skin diseases;

inflammatory diseases of the eye, blepharitis, dry eye syndrome and Sjogren

A syndrome; fibrosis of eyes.

The term "wound healing" refers to the complex process by which the skin (or another organ or tissue) repairs itself after injury. For example, in normal skin, the epidermis (the outermost layer) and dermis (the inner or deeper layer) exist in a steady state equilibrium, forming a protective barrier to the external environment. Once the protective barrier is breached, the normal (physiological) process of wound healing begins immediately. The classical model of wound healing is divided into three or four successive but overlapping stages: (1) hemostasis (which some authors do not consider to be a stage), (2) inflammation, (3) proliferation and (4) remodeling. In skin injury, a complex set of biochemical events occurs in a closely-fitting cascade to repair the injury. Within the first few minutes after injury, platelets adhere to the site of injury, become activated and aggregate (bind together), and subsequently activate the coagulation cascade, forming a clot of aggregated platelets in a network of cross-linked fibrin. This clot prevents active bleeding ("hemostasis"). During the inflammatory phase, bacteria and cellular debris are phagocytosed and removed from the wound by leukocytes. Platelet-derived growth factors (stored in the alpha granules of platelets) are released into the wound, which causes migration and division of cells during the proliferative phase. The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization and wound contraction. In "angiogenesis", vascular endothelial cells form new blood vessels. In "fibroplasia" and granulation tissue formation, fibroblasts grow by secreting collagen and fibronectin and form a new temporary extracellular matrix (ECM). At the same time, "re-epithelialization" of the epidermis occurs, wherein epithelial cells proliferate and "crawl" over the wound bed, providing coverage for new tissue. During wound contraction, myofibroblasts reduce the size of the wound by holding the wound edges and contracting using a mechanism similar to that in smooth muscle cells. As the action of the cell approaches completion, the unwanted cells undergo apoptosis. During maturation and remodeling, collagen is remodeled and rearranges along the tension line, while cells that are no longer needed are removed by apoptosis. However, this method is not only complex, fragile, and prone to interruption or failure, resulting in the formation of non-healing chronic wounds (one example includes diabetic wounds or ulcers, particularly diabetic foot ulcers). Factors that contribute to non-healing chronic wounds are diabetes, venous or arterial disease, infection and metabolic defects in the elderly.

The term "bone healing" or "fracture healing" refers to a proliferative physiological process in which the body promotes the repair of a fracture. During fracture healing, several stages of recovery promote proliferation and protection of the area surrounding the fracture and dislocation. The length of the procedure depends on the extent of the injury, and a two to three week margin is typically required for repair of most upper body fractures; any minor physical injury requires more than four weeks. The healing process is mainly determined by the "periosteum" (the connective tissue membrane covering the bone). Periosteum is a source of precursor cells that develop into "chondroblasts" and osteoblasts, which are essential for bone healing. Bone marrow (when present), endothelium, small blood vessels and fibroblasts are other sources of precursor cells.

In another embodiment, the compounds of table 1A or table 1B and pharmaceutically acceptable salts thereof as sGC stimulators are therefore useful for treating the following types of diseases, disorders or conditions, wherein stimulation of the process of wound or bone healing is desirable:

wound or ulcer healing in diabetic patients; improvement in microvascular perfusion; microvascular perfusion after injury improves or counteracts inflammatory reactions in perioperative care; anal fissure; diabetic ulcers; diabetic foot ulcers; bone healing; osteoclastic bone resorption and remodeling; and new bone formation.

The term "connective tissue" (CT) refers to an animal tissue that supports, connects or separates different types of tissues and organs of the body. It is one of four general classes of animal tissue, the remainder being epithelial, muscle and nerve tissue. Connective tissue is ubiquitous and is included in the central nervous system. It is located between other tissues. All CTs have three major components: matrix, fibers and cells, all of which are immersed in body fluids.

The term "connective tissue condition or disorder" refers to any disorder involving connective tissue abnormalities in one or more parts of the body. Certain disorders are characterized by overactivity of the immune system, resulting in tissue inflammation and systemic damage, and the replacement of normal tissue (e.g., of an organ) with connective tissue is common. Other diseases involve biochemical abnormalities or structural defects of the connective tissue itself. Some of these diseases are inherited and some cause is unknown.

When connective tissue diseases are of autoimmune origin, they are classified as "rheumatic disorders", "autoimmune rheumatic disorders" or "autoimmune collagen-vascular disorders".

In "autoimmune disorders," antibodies or other cells produced by the body attack the body's own tissues. Many autoimmune diseases affect connective tissue in various organs. In autoimmune diseases, inflammation and immune responses can lead to damage to connective tissue in the surrounding joints and in other tissues including vital organs such as the kidneys or organs of the gastrointestinal tract, the sac surrounding the heart (pericardium), the membrane covering the lungs (pleura), the mediastinum (disordered set of structures in the thorax surrounded by loose connective tissue, the large vessels containing the heart, the esophagus, the trachea, the phrenic nerve, the cardiac nerve, the thoracic duct, the thymus and the lymph nodes in the central thorax), and even the brain can be affected.

The term "fibrosis" as used herein refers to the accumulation of connective or fibrous tissue (scar tissue, collagen) in a certain organ or part of the body. If the fibrosis is from a single cell line, it is referred to as a "fibroma". Fibrosis occurs when the body attempts to repair and replace damaged cells and can therefore be a reactive, benign or pathological state. Physiological fibrosis is a process similar to scarring. When the tissue in question is repeatedly and continuously damaged, a pathological state is formed. Single lesions, even if severe, do not usually cause fibrosis. If the injury is repeated or continuous (as occurs in chronic hepatitis), the body attempts to repair the injury, but instead attempts to cause excessive accumulation of scar tissue. Scar tissue begins to replace the normal tissue of the organ that performs some function that the scar tissue cannot perform; it can also interfere with blood flow and limit blood supply to other cells. As a result, these other functional cells begin to die, forming more scar tissue. When this occurs in the liver, the blood pressure in the veins carrying blood from the intestine to the liver (portal vein) increases, resulting in a condition known as "portal hypertension".

The term "sclerosis" refers to the hardening of a tissue or structure or organ, which is generally flexible, usually replacing normal organ-specific tissue with connective tissue.

There are many types of fibrotic or fibrotic diseases, including but not limited to pulmonary fibrosis (idiopathic pulmonary fibrosis, cystic fibrosis), liver fibrosis (or "cirrhosis"), endomyocardial fibrosis, senile myocardial infarction, atrial fibrosis, mediastinal fibrosis, myelofibrosis (affecting the bone marrow), retroperitoneal fibrosis, progressive massive fibrosis (affecting the lung), nephrogenic fibrosis (affecting the skin), crohn's disease, articular fibrosis, peronias (affecting the penis), Dupuytren's (Dupuytren's) contracture (affecting the hands and fingers), some forms of adhesive capsulitis (affecting the shoulders).

There are many types of sclerosis or "sclerosing disease," including but not limited to Amyotrophic Lateral Sclerosis (ALS); atherosclerosis; focal segmental glomerulosclerosis and nephrotic syndrome; hippocampal sclerosis (affecting the brain); lichen sclerosis (a sclerosing disease of the connective tissue of the vagina and penis); cirrhosis (cirrhosis); multiple sclerosis or focal sclerosis (a disease that affects coordination); bone sclerosis (a disease in which bone density is significantly reduced); ear sclerosis (a disease affecting the ear); tuberous sclerosis (a rare genetic disease affecting multiple systems); primary sclerosing cholangitis (biliary cirrhosis); primary lateral sclerosis (progressive muscle weakness of voluntary muscles); and keloid scars.

The term "scleroderma" or "systemic sclerosis" or "progressive systemic scleroderma" refers to a condition involving scarring of joints, skin and internal organs and vascular abnormalities. Systemic sclerosis can sometimes occur in limited forms, for example sometimes affecting only the skin or mainly only certain parts of the skin or as CREST syndrome (where the surrounding area of the skin is involved, but not the trunk). The usual initial symptom of systemic sclerosis is swelling, followed by thickening and tightening of the skin at the end of the finger. In the "reynolds phenomenon", it is common for a finger to suddenly and temporarily become very pale and tingling or to become numb, painful or numb and painful.

The term "polymyositis" refers to muscle inflammation. The term "dermatomyositis" refers to inflammation of muscles that accompanies inflammation of the skin. The term "polychondritis" refers to inflammation of the cartilage.

The term "eosinophilic fasciitis" refers to a rare condition in which eosinophilic immune cells are released and cause inflammation and sclerosis of the "fascia," a tough fibrous tissue layer under the skin, above and between muscles. The fascia becomes painfully inflamed and swollen and gradually hardens in the arms and legs. As the skin of the arms and legs becomes increasingly hard, they become difficult to move and eventually become stuck in unusual positions. Sometimes, if the arm is involved, the person may develop carpal tunnel syndrome.

In another embodiment, specific conditions of diseases that can be treated and/or prevented by administering sGC stimulators in table 1A or table 1B and pharmaceutically acceptable salts thereof as sGC stimulators include, but are not limited to, the following types of diseases involving inflammation, autoimmunity or fibrosis (i.e., fibrotic diseases):

urogenital disorders: diabetic nephropathy; renal fibrosis and renal failure caused by chronic kidney disease or insufficiency; renal fibrosis and renal failure due to accumulation/deposition and tissue damage; nephrosclerosis; progressive hardening; glomerulonephritis; focal segmental glomerulosclerosis; nephrotic syndrome; prostatic hyperplasia; renal fibrosis; interstitial renal fibrosis;

diseases of the pulmonary system: pulmonary fibrosis; idiopathic pulmonary fibrosis; cystic fibrosis; progressive bulk fibrosis; progressive massive fibrosis affecting the lung;

diseases affecting the heart: endocardial myocardial fibrosis; senile myocardial infarction; atrial fibrosis; cardiac interstitial fibrosis; cardiac remodeling and fibrosis; cardiac hypertrophy;

diseases of the liver and related organs: liver cirrhosis or cirrhosis; cirrhosis associated with chronic liver disease; liver fibrosis; hepatic stellate cell activation; liver fibrous collagen and total collagen accumulation; necrotic inflammation and/or immunogenic liver disease; primary biliary cirrhosis; primary sclerosing cholangitis; other cholestatic liver diseases: diseases associated with granulomatous liver disease, liver malignancy, intrahepatic cholestasis of pregnancy, hepatitis, sepsis, drugs or toxins, graft versus host disease, liver transplantation, choledocholithiasis, cholangioma, pancreatic cancer, Miris' syndrome, AIDS cholangiopathy or parasites, schistosomiasis;

Digestive diseases or disorders: crohn's disease; ulcerative colitis; sclerosis of the gastrointestinal tract;

skin or eye diseases: renal fibrosis; keloid scars; fibrotic topical or skin diseases or conditions; fibrosis of dermis; scleroderma, skin fibrosis; hard spot disease; hypertrophic scars; moles; proliferative vitreoretinopathy; sarcoidosis; granuloma; ocular fibrosis;

diseases affecting the nervous system: amyotrophic Lateral Sclerosis (ALS); hippocampal sclerosis, Multiple Sclerosis (MS); focal hardening; primary lateral sclerosis;

bone disease; bone sclerosis;

hardening of the ear; other hearing diseases or disorders; hearing impairment, partial or total hearing loss; partial or total deafness; tinnitus; a noisy hearing loss;

other diseases involving autoimmunity, inflammation or fibrosis: scleroderma; local scleroderma or peripheral scleroderma; mediastinal fibrosis; fibrosing mediastinitis; myelofibrosis; retroperitoneal fibrosis; fibrosis of joints; peyronie's disease; dupu pure contracture; hardening lichens; some forms of adhesive capsulitis; atherosclerosis; nodular sclerosis; systemic hardening; polymyositis; dermatomyositis; polychondritis; bilirubin fasciitis; systemic lupus erythematosus or lupus erythematosus; myelofibrosis, myelofibrosis or myelofibroma; sarcoidosis; uterine fibroids; endometriosis.

In another embodiment, specific conditions of diseases that may be treated and/or prevented by administering sGC stimulators in table 1A or table 1B and pharmaceutically acceptable salts thereof as sGC stimulators include, but are not limited to: certain types of cancer; sickle cell disease; sickle cell anemia; metastasis of cancer; osteoporosis; gastroparesis; functional dyspepsia; diabetic complications; hair loss or loss of hair; diseases associated with endothelial dysfunction; neurological disorders associated with reduced nitric oxide production; argininosuccinic acid urine; neuromuscular diseases: duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), limb muscular dystrophy, distal myopathy, myotonic dystrophy type I and II, epidermal scapular dystrophy, autosomal and X-linked inherited idel-Dreifuss muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis and Spinal Muscular Atrophy (SMA).

In some embodiments, the present invention relates to a method of treating a disease, health condition, or disorder in a subject, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof, wherein the disease, health condition, or disorder is selected from one of the diseases listed above.

In another embodiment, the compounds of the invention may be delivered in the form of an implant device, such as a stent. Stents are reticulated "tubes" inserted into natural passages/conduits in the body to prevent or resist disease-induced, localized flow constriction. The term may also refer to a tube used to temporarily hold open such a natural catheter to allow surgical use.

Drug Eluting Stents (DES) are peripheral or coronary stents (stents) placed in stenotic, diseased peripheral or coronary arteries that slowly release drugs to block cell proliferation, usually smooth muscle cell proliferation. This prevents fibrosis and clot (thrombus) occlusion of the stented artery, i.e., the restenosis process. During angioplasty, stents are typically placed within the peripheral or coronary artery by an interventional cardiologist or interventional radiologist. Drugs commonly used in DES to block cell proliferation include paclitaxel or rapamycin analogues.

In some embodiments of the invention, the sGC stimulators of the invention may be delivered by a drug eluting stent coated with the sGC stimulators. Drug-eluting stents coated with the sGC stimulators of the present invention are useful for preventing stent restenosis and thrombosis in percutaneous coronary interventions. Drug-eluting stents coated with sGC stimulators according to the present invention may prevent smooth cell proliferation and aid revascularization and regeneration of endothelial tissue of the stented artery.

An alternative to percutaneous coronary intervention for the treatment of refractory angina caused by coronary occlusive disease is the method known as Coronary Artery Bypass Graft (CABG). CABG only provides ongoing procedural relief that is further complicated by the rapid development of graft atherosclerosis. Saphenous vein grafts are the most commonly used catheters in CABG procedures. Three major reasons that hinder the success of the long-term clinical trial of venous CABG are: graft atherosclerosis accelerates, incomplete endothelialization and thrombosis.

In some embodiments, sGC stimulators described herein can be used to prevent stealth graft failure during CABG. The compounds of the present invention may assist in the endothelialisation process and help prevent thrombosis. In this example, the sGC stimulating agent is delivered locally in the form of a gel.

The terms "disease," "symptom" and "disorder" are used interchangeably herein to refer to sGC, cGMP and/or NO-mediated medical or pathological disorders.

As used herein, the terms "subject" and "patient" are used interchangeably. The terms "subject" and "patient" refer to an animal (e.g., a bird, such as a chicken, quail or turkey or mammal), particularly a "mammal," including non-primates (e.g., cows, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice) and primates (e.g., monkeys, chimpanzees, and humans), and more particularly a human. In some embodiments, the subject is a non-human animal, such as a farm animal (e.g., a horse, cow, pig, or sheep) or a pet (e.g., a dog, cat, guinea pig, or rabbit). In some embodiments, the subject is a human.

The present invention also provides a method for treating one of the above-described diseases, disorders, and conditions in a subject, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof. Alternatively, the present invention provides the use of a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof, in the treatment of one of these diseases, disorders, and conditions in a subject in need thereof. The invention also provides methods of formulating or preparing a medicament for treating one of these diseases, disorders, and conditions, comprising using a compound of table 1A or table 1B, or a pharmaceutically acceptable salt thereof.

The term "biological sample" as used herein refers to an in vitro or ex vivo sample, including but not limited to cell cultures or extracts thereof; biopsy material obtained from a mammal or an extract thereof; blood, saliva, urine, feces, semen, tears, lymph, ocular fluid, vitreous humor, or other body fluids or extracts thereof.

"treating" with respect to a disorder or disease refers to reducing or eliminating the cause and/or effect of the disorder or disease. As used herein, the term "treatment" refers to reducing or ameliorating the progression, severity and/or duration of an sGC, cGMP and/or NO-mediated disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of the disorder (i.e., "controlling" rather than "curing" the disorder) resulting from administration of one or more treatments (e.g., one or more therapeutic agents, such as a compound or composition of the invention). In particular embodiments, the term "treating" refers to ameliorating at least one measurable physical parameter of an sGC, cGMP, and/or NO-mediated disorder. In other embodiments, the term "treating" refers to inhibiting the progression of sGC, cGMP, and/or NO-mediated disorders in the body, for example, by stabilizing discernible symptoms, or physiologically, for example, by stabilizing physical parameters, or both.

The term "prevention" as used herein refers to the pre-administration of a drug to avoid or prevent the appearance of one or more symptoms of a disease or disorder. One of ordinary skill in the medical arts recognizes that the term "prevention" is not an absolute term. In the medical field, it is to be understood that prophylactic administration of a drug to substantially reduce the likelihood or severity of a disorder or symptoms of a disorder is meant herein. The standard text in the field-Physician's Desk Reference uses the term "prevention" hundreds of times. As used herein, the terms "prevent", "preventing", "prevention" and "prevention" with respect to a disorder or disease refer to avoiding the onset, effect, symptom or progression of the disease or disorder before the disease or disorder is fully manifested.

In one embodiment, the methods of the invention are directed to the prevention, or "preempting", of a patient, particularly a human, predisposed (e.g., genetically predisposed) to having a sGC, cGMP, and/or NO-related disease, disorder, or symptom.

In other embodiments, the methods of the invention are directed to prophylaxis, or "preemptive," of a patient, particularly a human, at risk of having a sGC, cGMP, or NO-related disease, disorder, or symptom.

The compounds and pharmaceutical compositions described herein can be used alone or in combination therapy for the treatment or prevention of diseases or disorders mediated, modulated or affected by sGC, cGMP and/or NO.

The compounds and compositions disclosed herein are also useful in the veterinary treatment of companion, curious, and farm animals, including, but not limited to, dogs, cats, mice, rats, hamsters, gerbils, guinea pigs, rabbits, horses, pigs, and cattle.

In other embodiments, the invention provides methods of stimulating sGC activity in a biological sample comprising contacting the biological sample with a compound or composition of the invention. The use of sGC stimulators in biological samples is useful for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, biological assays and biological sample storage.

Combination therapy

The compounds and pharmaceutical compositions described herein may be used in combination with one or more additional therapeutic agents. For combination therapy with more than one active agent, when the active agents are separate dosage formulations, the active agents may be administered alone or in combination. In addition, one component may be administered before, simultaneously with, or after the other agent.

When co-administered with other agents, for example when co-administered with another analgesic drug, the "effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by one of skill in the art depending on the condition of the subject, the type of disorder to be treated and the amount of compound used. In the case where the amount is not explicitly indicated, an effective amount should be employed. For example, the compounds described herein may be administered to a subject in a dosage range of about 0.01 to about 10,000mg/kg body weight/day, about 0.01 to about 5000mg/kg body weight/day, about 0.01 to about 3000mg/kg body weight/day, about 0.01 to about 1000mg/kg body weight/day, about 0.01 to about 500mg/kg body weight/day, about 0.01 to about 300mg/kg body weight/day, about 0.01 to about 100mg/kg body weight/day.

When "combination therapy" is used, a first amount of a compound in table 1A or table 1B, or a pharmaceutically acceptable salt thereof, and a second amount of an additional suitable therapeutic agent may be used to achieve an effective amount.

In one embodiment of the invention, the compound of table 1A or table IB and the additional therapeutic agent are each administered in an effective amount (i.e., if each amount alone is a therapeutically effective amount). In another embodiment, the compound of table 1A or table 1B and the additional therapeutic agent are each administered in an amount that does not provide a therapeutic effect (sub-therapeutic dose). In another example, the compounds of table 1A or table 1B may be administered in an effective amount, while the additional therapeutic agent is administered in a sub-therapeutic dose. In another example, a compound of table 1A or table 1B can be administered in a subtherapeutic dose, while an additional therapeutic agent, such as a suitable cancer therapeutic agent, is administered in an effective amount.

As used herein, the terms "combination" or "co-administration" are used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). The use of the terms does not limit the order in which therapeutic methods (e.g., prophylactic and/or therapeutic agents) are administered to a subject.

Co-administration includes administering the first and second amounts of the compound in a substantially simultaneous manner, e.g., in a single pharmaceutical composition, e.g., a capsule or tablet having a fixed ratio of the first and second amounts, or multiple, separate capsules or tablets. In addition, such co-administration also includes the sequential use of each compound in any order. When co-administration involves separate administration of a first amount of a compound of table 1A or table 1B and a second amount of an additional therapeutic agent, the compounds are administered close enough in time to have the desired therapeutic effect. For example, the time period between each administration that can produce the desired therapeutic effect can range from a few minutes to a few hours, and the properties of each compound, such as potency, solubility, bioavailability, plasma half-life and kinetic characteristics, can be considered. For example, the compound of table 1A or table 1B and the second therapeutic agent can be administered in any order within about 24 hours of each other, within about 16 hours of each other, within about 8 hours of each other, within about 4 hours of each other, within about 1 hour of each other, or within about 30 minutes of each other.

More specifically, a first treatment (e.g., a prophylactic or therapeutic agent, such as a compound described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second treatment (e.g., a prophylactic or therapeutic agent, such as an anti-cancer agent) to a subject.

Examples of other therapeutic agents that may be combined with the compounds herein administered alone or in the same pharmaceutical composition include, but are not limited to:

(1) endothelial Derived Release Factor (EDRF);

(2) NO donors, such as nitrosothiols, nitrites, schidmamines, NONO nucleophiles, N-nitrosamines, N-hydroxynitrosamines, nitrosoimines, nitrotyrosines, diazabicyclo, oxatriazole 5-imines, oximes, hydroxylamines, N-hydroxyguanidines, hydroxyureas or furanoses. Some examples of these types of compounds include: glycerol trinitrate (also known as GTN, nitroglycerin, and trinitroglycerin), the nitrate ester of glycerol; sodium Nitroprusside (SNP), in which a nitric oxide molecule is coordinated to the iron metal forming a square bipyramid complex; 3-morpholinodiimine (SIN-1), a zwitterionic compound formed by a combination of morpholine and xidecmine; S-nitroso-N-acetylpenicillamine (SNAP), N-acetylated amino acid derivatives with nitrosothiol functionality; diethylenetriamine/NO (DETA/NO), a nitric oxide compound covalently linked to diethylenetriamine; and NCX4016, the m-nitromethylphenyl ester of acetylsalicylic acid. More specific examples of some of these classes of NO donors include: classical nitrovasodilators such as organic nitrates and nitrites including nitroglycerin, amyl nitrite, isosorbide dinitrate, isosorbide 5-nitrate and nicorandil; isosorbide ester

FK409 (NOR-3); FR144420 (NOR-4); 3-morpholinounsaturated imine chlorohydrate dichloride ("SIN-1"); S-nitroso-N-acetylpenicillamine ("SNAP"); AZD3582(CINOD lead compound), NCX4016, NCX701, NCX1022, HCT1026, NCX1015, NCX950, NCX1000, NCX1020, AZD4717, NCX1510/NCX1512, NCX2216 and NCX404 (all available from NicOx s.a.), S-nitrosoglutathione (GSNO), sodium nitroprusside, S-nitrosoglutathione monoethyl ester (GSNO-ester), 6- (2-hydroxy-1-methyl-nitrosohydrazino) -N-methyl-1-hexylamine (NOC-9) or diethylamine NONO nucleophilic complex. Nitric oxide donors are also described in U.S. Pat. Nos. 5,155,137,5,366,997,5,405,919,5,650,442,5,700,830,5,632,981,6,290,981,5,691,423,5,721,365,5,714,511,6,511,911 and 5,814,666, Chrysselis et al (2002) journal of pharmaceutical chemistry, 45: 5406-9 (e.g. NO donors 14 and 17), and nitric oxide donors in pharmaceutical and biological research, authors: rong Wang, Dingwei Wei and Guzuoqiang; willi, 2005;

(3) other substances that increase the concentration of cGMP, such as protoporphyrin IX, arachidonic acid and phenylhydrazine derivatives;

(4) nitric oxide synthase substrate: for example, N-hydroxyguanidine base analogs such as N [ G ] -hydroxy-L-arginine (NOHA), 1- (3, 4-dimethoxy-2-chlorobenzylideneamino) -3-hydroxyguanidine and PR5(1- (3, 4-dimethoxy-2-chlorobenzylideneamino) -3-hydroxyguanidine); l-arginine derivatives (e.g., homoArg, homoNOHA, N-t-butoxy and N- (3-methyl-2-butenyl) oxy-L-arginine, canavanine, epsilon-myristic acid, agmatine, hydroxy-martin and L-tyrosyl-L-arginine); N-alkyl-N ' -hydroxyguanidines (e.g., N-cyclopropyl-N ' -hydroxyguanidine and N-butyl-N ' -hydroxyguanidine), N-aryl-N ' -hydroxyguanidines (e.g., N-phenyl-N ' -hydroxyguanidine and its derivatives para-substituted with-F, -Cl, -methyl, -OH substituents, respectively); guanidine derivatives, such as 3- (trifluoromethyl) propylguanidine; other materials summarized by Cali et al (2005, pharmaceutical chemistry lesson 5: 721-;

(5) Compounds that enhance eNOS transcription: such as those described in WO 02/064146, WO 02/064545, WO 02/064546 and WO 02/064565, and corresponding patent documents such as US2003/0008915, US2003/0022935, US2003/0022939 and US 2003/0055093. Including other eNOS transcription enhancers described in US20050101599 (e.g., 2-difluorobenzo [1,3 ]]Dioxole-5-carboxylic acid indan-2-ylamide and 4-fluoro-N- (indan-2-yl) -benzamide) and seofine-avanta compounds AVE3085 and AVE9488(CA accession No. 916514-70-0;

et al, journal of thrombosis and hemostasis, 2005; volume 3, makeup version 1: digest number P1487);

(6) NO-independent heme-independent sGC stimulators, including but not limited to: BAY58-2667 (see patent publication DE19943635)

HMR-1766 (Atxiguat sodium, see patent publication WO2000002851)

(2- (4-chloro-phenylsulfonylamino) -4, 5-dimethoxy-N- (4- (thiomorpholine-4-sulfonyl) -phenyl) -benzamide (see patent publications DE19830430 and WO2000002851)

And HMR-1069 (cenofil-amphetate).

(7) Heme-dependent sGC stimulators, including but not limited to:

YC-1 (see patent publications EP667345 and DE19744026)

Riociguat (BAY 63-2521, commercial product, described in DE19834044)

Riciguat (BAY 60-4552, described in WO 2003095451)

Vicigua (BAY 1021189, temporarily backed up to Liocigua),

BAY 41-2272 (described in DE19834047 and DE19942809)

BAY 41-8543 (described in DE19834044)

Itraciguat (described in WO 2003086407)

CFM-1571 (see patent publication WO2000027394)

A-344905, its acrylamide analog A-350619 and aminopyrimidine analog A-778935.

Compounds disclosed in the following publications: US20090209556, US8455638, US20110118282(WO2009032249), US20100292192, US20110201621, US7947664, US8053455(WO2009094242), US20100216764, US8507512, (WO2010099054), US20110218202(WO2010065275), US20130012511(WO2011119518), US20130072492(WO2011149921), US20130210798(WO2012058132) and in tetrahedrons (2003), 44 (48): other compounds disclosed in 8661-8663.

(8) Compounds that inhibit cGMP degradation, for example:

PDE5 inhibitors, e.g. sildenafil

And other related drugs, such as avanafil, rotinafil, milonafil, sildenafil citrate

Tadalafil (R)

Vardenafil

And udenafil; alprostadil and dipyridamole; PF-00489791; PDE9 inhibitors, such as PF-04447943;

(9) Calcium channel blockers such as:

dihydropyridine calcium channel blockers: amlodipine (amlodipine), aranidipine (aranidipine), azelnidipine (carbopol), barnidipine (sea wave), benidipine (any), cilnidipine (ataractic, cinalone, cissa), clevidipine (clevidipine butyrate), diltiazem, efonidipine (Lidell), felodipine (boedipine), lacidipine (moxon, lacitinib), lercildipine (lercanidipine), manidipine (Carlotide, meldipine), nicardipine (nicardipine, CardenSR), nifedipine (reserpine, heartache), nilvadipine (nilvatinib), nimodipine (nimotron), nisoldipine (Bemcard, nisoldipine, film-coated controlled release tablets), nildipine (Gadifu, nitrendipine, beverine), and lacidipine (acarpine), isradipine (lomicron);

phenylalkylamine calcium channel blockers: verapamil (Kalan, verapamil)

Gelopamid (galopamid, D600);

diltiazem species: diltiazem (sweet heart);

non-selective calcium channel inhibitors, such as: imazadil, bepridil and fluspirilen, fentanyl;

(10) Endothelin Receptor Antagonist (ERA): for example, the dual (ETA and ETB) endothelin receptor antagonists bosentan

(ii) a Sitaxsentan, to

Naming for sale; ambrisentan is sold in the United states as

(ii) a The dual/non-selective endothelin antagonist aivlon-1 entered clinical trials in 2008;

(11) prostacyclin derivatives or analogs: such as prostacyclin (prostaglandin I)₂) Epoprostenol (synthetic prostacyclin, or

Sales); treprostinil

Iloprost, iloprost

Iloprost, iloprost

(ii) a In oral and inhaled form

In development; beraprost, oral prostaglandins in japan and korea;

(12) antihyperlipidemic drugs, such as: bile acid sequestrants (e.g., choline amine, colestipol, and colesevelam); statins, such as atorvastatin, simvastatin, lovastatin, fluvastatin, pitavastatin, rosuvastatin and pravastatin; cholesterol absorption inhibitors, such as ezetimibe; other lipid lowering agents, such as ethyl pentacosate, ethyl omega-3-oate, ridol; fibric acid derivatives such as brefibrate, bezafibrate, clinofibrate, gemfibrozil, rofibrate, bifilxofen, fenofibrate, ciprofibrate, choline fenofibrate; nicotinic acid derivatives such as acipimox and niacin; a combination of statins, niacin, an intestinal cholesterol absorption inhibiting supplement (ezetimibe, etc.) and fibric acids; antiplatelet therapeutic agents, such as clopidogrel hydrogensulfate;

(13) Anticoagulants, of the following types:

coumarin (vitamin K antagonist):

(Kemaiding), the mainTo be used in the united states and united kingdom;

and

mainly used in other countries;

；

heparin and derived substances, such as: heparin; low molecular weight heparin, fondaparinux and idazin;

direct thrombin inhibitors, such as: argatroban, lopidine, bivalirudin and dabigatran; ximei gallon

No U.S. approval;

tissue plasminogen activators for dissolving clots and for removing arterial occlusions, such as alteplase;

(14) antiplatelet drugs: for example thienopyridines such as lopepipeptide and ticlopidine; dipyridamole aspirin;

(15) ACE inhibitors, for example of the following types:

drugs containing mercapto groups, e.g. captopril

A first ACE inhibitor and zofenopril;

medicaments containing dicarboxylic acid salts, e.g. enalapril (enalapril @)

) (ii) a Ramipril (ramipril/cardiodamid/remasu |)

) (ii) a Quinolones

Perindopril (Biofamarl @)

) (ii) a Lisinopril (Castanel/Lepril/Norwalk/lisinopril

) And benazepril

；

Phosphonic acid-containing drugs, such as: fosinopril;

naturally occurring ACE inhibitors, such as: casketoni and lettoni, which are decomposition products of casein and whey naturally produced upon ingestion of a milk product, particularly cultured milk; the lactic acid tripeptides Val-Pro-Pro and Ile-Pro-Pro produced by the probiotic Lactobacillus helveticus or casein also have ACE inhibitory and antihypertensive functions;

Other ACE inhibitors, such as alaceprednol, delapril, cilazapril, imidapril, trandolapril, temocapril, moexipril, spirapril,

(16) auxiliary oxygen therapy;

(17) beta blockers, such as the following types:

non-selective drugs:

(with other alpha-blocking activity),

(with other alpha-blocking activity),

，

(having aIntrinsic sympathetic activity),

(with intrinsic sympathetic activity), oxerlotinolol, acebutolol, sotalol, mepindolol, celiprolol, arotinolol, terbutalol, sulfamolol, nipropranolol,

and

；

selective beta₁-receptor blockers:

(with intrinsic sympathetic activity),

dobutamine hydrochloride, elsodipine maleate, carvedilol, talinolol, esmolol, metoprolol and

；

selective beta₂-receptor blockers:

(weak alpha-adrenergic agonist activity);

(18) antiarrhythmic drugs, of the following types:

type I (sodium channel blocker): quinidine, lidocaine, phenytoin, propafenone;

type III (potassium channel blocker): amiodarone, dofetilide, sotalol;

form V: adenosine, digoxin;

(19) diuretics, for example: thiazide diuretics, such as chlorothiazide, chlorothiadone and hydrochlorothiazide, fluazifozide, cyclopenthiazide, methyltriazide, polythiazide, quinethione, cyproteam, metolazone, indapamide, cilastarin; loop diuretics such as furosemide and toresistine; potassium sparing diuretics such as amiloride, spironolactone, potassium canrenoate, eplerenone and triamterene; combinations of these agents; other diuretics, such as acetazolamide and capecitabine;

(20a) Direct acting vasodilators such as hydralazine hydrochloride, diazoxide, sodium nitroprusside, catalazine; other vasodilators, such as isosorbide dinitrate and isosorbide 5-nitrate;

(20b) exogenous vasodilators, such as:

·

adenosine agonists, primarily used as antiarrhythmics;

alpha blocker (blocking the vasoconstrictive action of epinephrine):

alpha-1-adrenergic receptor antagonists, e.g. prazosin, indolizines, urapidil, bunazosin, terazosin, doxazosin

Atrial Natriuretic Peptide (ANP);

ethanol;

histamine inducers which cause the action of the supplementary proteins C3a, C4a and C5a by triggering the release of histamine from mast cells and basophils;

tetrahydrocannabinol (THC), a principal active chemical of cannabis, with a mild vasodilating effect;

papaverine, an alkaloid found in poppy flower; b

(21) Bronchodilators: there are two main types of bronchodilators, β₂Agonists and anticholinergic agents, as follows:

·β₂agonist(s):

or salbutamineAlcohol (Universal brand: Vantolin) and

short-acting beta that is a rapid relief of COPD symptoms₂An agonist. Long acting beta₂Agonists (LABA) such as

And

；

anticholinergic agents:

is the most widely used short-acting anticholinergic drug.

Are the most commonly used long-acting anticholinergic agents in COPD;

·

bronchodilators and phosphodiesterase inhibitors;

(22) corticosteroid: for example beclomethasone, methylprednisolone, betamethasone, prednisone, prednisolone, triamcinolone acetonide, dexamethasone, fluticasone, flunisolide and hydrocortisone, and corticosteroids such as budesonide;

(23) dietary supplements, for example: omega-3 oils; folic acid, niacin, zinc, copper, Korean red ginseng, ginkgo biloba, pine bark, tribulus terrestris extract, arginine, oat, epimedium, maca root, ferrieritine, saw palmetto and Swedish pollen; vitamin C, vitamin E, vitamin K2; testosterone supplements, testosterone transdermal patches; clavulanic acid, naltrexone, brimordane (formerly PT-141), melanotan II, hmadex-K; raylex: proprietary mixtures/compositions of the natural ingredients L-arginine aspartate and pycnogenol;

(24) PGD2 receptor antagonists including, but not limited to, those described in U.S. published applications US200200222218, US20010051624 and US20030055077, PCT published applications WO9700853, WO9825919, WO03066046, WO03066047, WO03101961, WO03101981, WO04007451, WO0178697, WO04032848, WO03097042, WO03097598, WO03022814, WO03022813 and WO04058164, european patent applications EP945450 and EP944614 having PGD2 antagonistic activity, and Torisu et al, 2004, bio-organic and pharmaceutical chemistry 14: 4557; torisu et al, 2004, bio-organic and pharmaceutical chemistry 14: 4891 and Torisu et al, 2004, bio-organic and pharmaceutical chemistry 2004, 12: 4685;

(25) Immunosuppressive agents, such as cyclosporine (cyclosporin a,

) Tacrolimus (FK-506,

) Rapamycin (sirolimus,

) And other FK-506 type immunosuppressants, and mycophenolate esters, e.g. mycophenolate esters

；

(26) Non-steroidal anti-asthmatics, e.g. beta₂Receptor agonists (e.g. terbutaline, metagonine, fenoterol, isolauryl alcohol, salbutamol, salmeterol, kuh-seng alcohol and pirbuterol) and beta₂Combinations of-agonists and corticosteroids (e.g. salmeterol-fluticasone)

Formoterol-budesonide

) Theophylline, cromolyn sodium, nedocromil, atropine, ipratropium bromide, leukotriene biosynthesis inhibitor (qi circulation, BAY 1005);

(27) non-steroidal anti-inflammatory drugs (NSAIDs), such as propionic acid derivatives (e.g., alisprofen, benprolene, butofenac, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miprolofen, naproxen, oxaprozin, pyrrolprofen, pranoprofen, suprofen, celecoxib, and tioprofen), acetic acid derivatives (e.g., indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclorac, fentiazac, furofenac, ibufenac, isoxac, oxydiphosphate, sulindac, thiopinac, tolmetin, qidometacin, and zomepirac), fenamic acid derivatives (e.g., flufenamic acid, meclofenamic acid, mefenamic acid, niflumic, and tolfenamic acid), biphenylcarboxylic acid derivatives (e.g., diflunisal and flufenisal), oxicams (e.g., isoxicam, piroxicam, sudoxicam and terbacil), salicylates (e.g. acetylsalicylic acid and sulfasalazine) and pyrazolones (e.g. apalone, pirfenidone, flupraziquantel, morphinolone, oxypetasone and phenylbutazone).

(28) Cyclooxygenase-2 (COX-2) inhibitors, such as celecoxib

Rofecoxib

Valdecoxib, etoricoxib, parecoxib and rocheib; (opioid analgesics such as codeine, fentanyl, hydromorphone, levorphanol, meperidine, methadone, morphine, oxycodone, oxymorphone, propoxyphene, buprenorphine, butorphanol, tezomib, nepafen and pentazocine; and

(29) antidiabetic agents, e.g. insulin and insulin mimeticsMimetics, sulfonylureas (e.g. glipizide, glyburide, glipizide, gliclazide, gliquidone, glimepiride, glipide, tolbutamide, chlorpropamide, acetohexamide, tolazamide), biguanides, e.g. metformin

Alpha-glucosidases (e.g. acarbose, epalrestat, voglibose, miglitol), thiazolidinone compounds, e.g. rosiglitazone

Troglitazone

Ciglitazone, pioglitazone

And englitazone; insulin sensitizers such as pioglitazone and rosiglitazone; insulin secretagogues such as repaglinide, nateglinide and mitiglinide; incretin mimetics such as isapeptide and liraglutide; amylin analogs such as pramlintide; glucose lowering agents, such as chromium picolinate (photosynthetic biotin); dipeptidyl peptidase IV inhibitors such as sitagliptin, vildagliptin, saxagliptin, alogliptin and linagliptin; vaccines currently under development for the treatment of diabetes; AVE-0277, alum-GAD, BHT-3021, IBC-VS 01; cytokine targeted therapeutic drugs developed for the treatment of diabetes, such as anakinra, canamab, diacerein, gemumab, LY-2189102, MABP-1, GIT-027; drugs developed for the treatment of diabetes:

(30) HDL cholesterol increasing agent such as Anacetrapib, MK-524A, CER-001, DRL-17822, Dacetrapib, JTT-302, RVX-000222, TA-8995;

(31) slimming drugs, e.g. methamphetamine hydrochloride, bupropion hydrochloride

Benz butylamine

Benzphetamine hydrochloride

Phendimetrazine tartrate

Mazindol

Orlistat

Sibutramine hydrochloride monohydrate

Rimonabant

Amfepramone, chromium picolinate, RM-493, TZP-301; combinations such as phentermine/topiramate, bupropion/naltrexone, sibutramine/metformin, amphetamineNon-tardone SR/zonisamide SR, salmeterol, xinafoate/fluticasone propionate; lorcaserin hydrochloride, phentermine/topiramate, bupropion/naltrexone, cetrizatriptan, exenatide, KI-0803, liraglutide, metformin hydrochloride, sibutramine/metformin, 876167, ALS-L-1023, bupropion SR/zonisamide SR, CORT-108297, canaifene, chromium picolinate, GSK-1521498, LY-377604, metreleptin, ornipide, P-57AS3, PSN-821, salmeterol sulfate/fluticasone propionate, sodium tungstate, growth hormone (recombinant), TM-30339, TTP-435, tesamolin, tesofensine, virofibrate, zonisamide, BMS-830216, ALB-127158, AP-HX-1030, ATD-105, ATD-2820, AZD-8329, polarenib and milaloirane (Bereox), CP-404, HPP-404, ISIS-FGFR4Rx, insulin, KD-3010PF, 05212389, PP-1420, PSN-842, peptide YY3-36, resveratrol, S-234462; s-234462, color bit meter (Sobetirome), TM-38837, tetrahydrovancomycin, ZYO-1, beta-lapachone;

(32) Angiotensin receptor blockers such as losartan, valsartan, candesartan cilexetil, eprosartan, irbesartan, telmisartan, olmesartan medoxomil, azinazole alkyl esters;

(33) renin inhibitors, such as aliskiren semipseudomonas;

(34) centrally acting alpha-2-adrenoceptor agonists such as methyldopa, clonidine, guanfacine;

(35) adrenergic neuron blocking agents, such as guanethidine, guanadine;

(36) imidazoline I-1 receptor agonists, such as the phosphate bicyclic phosphate and the hydrochloric acid Moconidine hydrate;

(37) aldosterone antagonists such as spironolactone and eplerenone;

(38) potassium channel activators such as pinadil;

(39) dopamine D1 agonists, such as meflufene; other dopamine agonists, such as bopomine, docetaxel and polycarbobamine;

(40)5-HT2 antagonists, such as ketotannins;

(41) drugs currently under development for the treatment of arterial hypertension:

(42) vasopressin antagonists, such as tolvaptan;

(43) calcium channel sensitizers, such as levosimendan or agonists, such as nicorandil;

(44) PDE-3 inhibitors such as amiloride, fenpyrazamine, enoximone, vesnarinone, pimobendan, olprinone;

(45) Adenylate cyclase activators such as dopidine hydrochloride;

(46) inotropic agents, such as digoxin and mie toxin; metabolic cardiotonic agents such as ubidecarenone; brain-like peptides, such as nesiritide;

(47) drugs currently under development for the treatment of heart failure:

(48) drugs currently developed for the treatment of pulmonary hypertension

(49) Drugs currently developed for the treatment of female sexual disorders

(50) Drugs for treating erectile dysfunction, such as alprostadil, avidine, phentolamine mesylate, vigilant, alprostadil;

(51) drugs currently developed for the treatment of male sexual dysfunction:

(51) drugs developed for the treatment of sleep apnea:

(52) drugs currently developed for metabolic syndrome:

(53) the weight-losing medicine comprises the following components:

(54) drugs for the treatment of alzheimer's disease: cholinesterase inhibitors, e.g., for mild to moderate Alzheimer's disease, include

(galanthamine) is added to the composition,

(Rivastigmine) and

(donepezil) in the form of a pharmaceutically acceptable salt,

(tacrine);

(memantine), N-methyl D-aspartate (NMDA) antagonists and

for the treatment of moderate to severe Alzheimer's disease; vitamin E (antioxidant).

(55) An antidepressant: tricyclic antidepressants, e.g. amitriptyline

Desipramine

Imipramine

Amoxicillin

Nortriptyline; selective 5-hydroxytryptamine reuptake inhibitors (SSRI's), such as paroxetine

Fluoxetine

Of sertraline

And lemon pulan

(ii) a Others such as doxepin

And trazodone

(ii) a SNRIs (e.g., venlafaxine and reboxetine); dopaminergic antidepressants (e.g., bupropion and aminopeptides).

(56) Neuroprotective agents: such as memantine, levodopa, bromocriptine, agogat, talipexole, pramipexole, cabergoline, neuroprotective agents currently under investigation, including anti-apoptotic drugs (CEP 1347 and CTCT346), lazaroy, bioenergetic therapy, anti-glutamatergic drugs and dopamine receptors. Other clinically evaluated neuroprotective agents such as the monoamine oxidase B inhibitors selegiline and rasagiline, dopamine agonists and the complex I mitochondrial enhancer coenzyme Q10.

(57) Antipsychotic agents: such as ziprasidone (ziprasidone)^TM) Risperidone (risperidone)^TM) And olanzapine (olanzapine)^TM)。

(58) NEP inhibitors such as sabotart, Opatraga.

(59) Methylene Blue (MB)

Reagent kit

The compounds and pharmaceutical formulations described herein may be contained in a kit. Kits may include a single or multiple doses of two or more reagents, each of which is packaged or formulated separately, or packaged in combination or formulated as a single or multiple doses of two or more reagents. Thus, one or more reagents may be present in a first container, and the kit may optionally include one or more reagents in a second container. The container or containers are placed within a package, and the package may optionally include instructions for administration or dosing. The kit may include additional components, such as a syringe or other means for administering the agent as well as a diluent or other means for formulating the agent. Thus, the kit may comprise: a) a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier, vehicle or diluent; and b) a container or package. The kit may optionally include instructions describing a method of using the pharmaceutical composition in one or more methods described herein (e.g., preventing or treating one or more diseases and disorders described herein). The kit may optionally comprise a second pharmaceutical composition comprising one or more additional medicaments, pharmaceutically acceptable carriers, vehicles or diluents for co-therapeutic use as described herein. Pharmaceutical compositions comprising a compound described herein and a second pharmaceutical composition contained in the kit may optionally be combined in the same pharmaceutical composition.

The kit comprises a container or package for containing the pharmaceutical composition and may also comprise a separate container, such as a separate bottle or a divided foil packet. The containers may be, for example, paper or cardboard boxes, glass or plastic bottles or cans, resealable bags (e.g., to hold a filling of tablets that may be placed in different containers), or blister packs, wherein each dose is pressed out of the pack according to a treatment regimen. Multiple containers may be used together in a single package to sell a single dosage form. For example, the tablets may be contained in a bottle which in turn is contained within a box.

An example of a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, etc.). Blister packs are generally made of a relatively rigid material covered with a foil, preferably of a transparent plastic material. During packaging, recesses are formed in the plastic foil. These recesses may be of the size and shape of the individual tablets or capsules to be packaged, or may be of the size and shape to accommodate a plurality of tablets and/or capsules to be packaged. Next, tablets or capsules are placed in the recesses, respectively, and a sheet of relatively rigid material is sealed onto the plastic foil at the surface of the foil opposite to the direction in which the recesses are formed. Thus, the tablets or capsules are sealed individually or collectively in the recesses between the plastic foil and the sheet, as required. Preferably, the strength of the sheet is such that the tablet or capsule can be removed from the blister pack by manually applying pressure on the recesses, thereby forming openings in the sheet at the location of the recesses. The tablet or capsule can then be removed through the opening.

It may be advantageous to provide a written memory aid containing information and/or instructions about the physician, pharmacist or subject when to take the medication. The "daily dose" may be a single tablet or capsule or several tablets or capsules ingested on a given day. When the kit contains separate compositions, a daily dose of one or more of the compositions of the kit may consist of one tablet or capsule, while a daily dose of another one or more of the compositions of the kit may consist of a plurality of tablets or capsules. The kit may take the form of a dispenser to dispense the daily doses one at a time in the order of their intended use. The dispenser may be provided with a memory aid to further facilitate compliance with the protocol. An example of such a memory aid is a mechanical counter which indicates the number of daily doses that have been dispensed. Another example of such a memory aid is a battery powered microchip memory coupled to a liquid crystal reading device or an audible reminder signal, for example, to read the date the last daily dose has been extracted and/or to remind the date the next dose is extracted.

Examples

All references provided in the examples are incorporated herein by reference. As used herein, all abbreviations, symbols and conventions are consistent with those used in the contemporary scientific literature. See, e.g., "ACS style guidelines," authored by Janet s. Author and editing manual ", 2 nd edition, washington d.c.: the american chemical society, 1997, the entire contents of which are incorporated herein by reference.

Example 1 Synthesis of Compounds in Table IA or Table IB

General method A

Step 1:

diketone enolate formation:to a solution of ketone a in THF cooled to-78 ℃, LiHMDS (e.g., 0.9 eq, 1.0M in toluene) was added dropwise via syringe. The reaction was warmed to 0 ℃ and diethyl oxalate (1.2 eq) was added. The reaction was warmed to room temperature and stirred at that temperature until judged complete (e.g., using TLC or LC/MS analysis). Once the reaction is complete (reaction time is typically 45 minutes), the product diketoenolate B is used "directly" in step 2, i.e. the cyclisation step, without any further purification.

Step 2:

pyrazole formation:diketoenolate B was diluted with ethanol and HCl (e.g., 3 equivalents, 1.25M in ethanol) and aryl hydrazine hydrate (e.g., 1.15 equivalents) were added continuously. The reaction mixture is heated to 70 ℃ and stirred at this temperature until cyclization is deemed complete (e.g., by LC/MS analysis, typically 30 minutes). Once complete, the reaction mixture is carefully treated with solid sodium bicarbonate (e.g., 4 equivalents) and diluted with dichloromethane and water. The layers were separated and the aqueous layer was further diluted with water and then extracted three times with dichloromethane. The combined organics were washed with brine, over MgSO ₄Dried, filtered and concentrated in vacuo. Then a suitable gradient of EtOAc in hexane was used, passing through the SiO₂And (4) carrying out chromatography purification to obtain pyrazole C.

And step 3:

amidine formation:to NH cooled to 0 DEG C₄AlMe is added dropwise via syringe to a suspension of Cl (e.g. 5 equivalents) in toluene₃(e.g., 5 equivalents, 2.0M in toluene). The reaction was warmed to room temperature and stirred at that temperature until no more bubbles were observed. Pyrazole C is added to the reaction mixture in one portion, heated to 110 ℃, and stirred at that temperature until the reaction is judged to be complete (e.g., using TLC or LC/MS analysis). Once complete, the reaction was cooled, treated with excess methanol, and stirred vigorously at room temperature for 1 hour. The viscous slurry was filtered and the resulting solid filter cake was washed with methanol. The filtrate was concentrated in vacuo and the resulting solid was resuspended in ethyl acetate: isopropanol-5: 1 in a solvent mixture. The reaction was further treated with saturated sodium carbonate solution, stirred for 10 minutes, and then the layers were separated. The aqueous layer was washed with ethyl acetate: a solvent mixture (3 ×) of isopropanol ═ 5:1 was extracted and the combined organics were washed with brine. The organics were further treated with MgSO₄Drying, filtering and removing the solvent in vacuum. The product, amidine D, was used directly in the next step without further purification.

And 4, step 4:

pyrimidinone formation:amidine D was suspended in ethanol and stirred vigorously at 23 ℃ to promote complete solvation. Sodium 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (e.g. 3 equivalents) was added and the flask was equipped with a reflux condenser. The reaction was placed in a pre-heated oil bath maintained at 90 ℃ and stirred until complete consumption of starting material was observed by LC/MS (reaction time typically 1 hour). The reaction is cooled to 23 ℃ and the reaction mixture is acidified with HCl (e.g., 3 equivalents, 1.25MEtOH solution). The mixture was stirred for 30 minutes and most of the solvent was removed in vacuo. The reaction was resuspended in ether and water (1:1 mixture) and the resulting slurry was stirred for 20 minutes. The suspension was filtered under vacuum and the solid cake was washed with additional water and ether and dried under high vacuum overnight. The pyrimidinone E obtained is used directly in the subsequent step without further purification.

General method B

A solution of the amino nucleophile (3 equiv.), triethylamine (10 equiv.) and intermediate-1A (1 equiv.) was stirred in dioxane and water (2:1 ratio) at 90 deg.C until complete consumption of starting material was observed by LC/MS. The solution was diluted with 1N aqueous hydrochloric acid and dichloromethane. The layers were then separated and the aqueous layer was extracted with dichloromethane. The organics were combined, dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification to give the desired product.

General method C

To a mixture of intermediate-2 (which was described in the previously published patent application WO2012/3405a 1; 1 eq) and carboxylic acid (1.1 eq) in N, N-dimethylformamide was added successively triethylamine (4 eq) and a 50% solution of propylphosphonic anhydride (T3P, 1.4 eq) in ethyl acetate. The reaction was heated to 80 ℃ for 24 hours and then the reaction was diluted with water and 1N hydrochloric acid solution. The diluted reaction was extracted with dichloromethane and then ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Purification to give the desired product.

Synthesis of intermediate-1A

A mixture of 5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -pyrimidin-4-ol (intermediate-5A; produced by general method A using 1- (isoxazol-3-yl) ethanone in step 1 and 2-fluorobenzylhydrazine in step 2, 11.5g, 32.4mmol, 1 eq) and phosphoryl trichloride (60.3mL, 647mmol, 20 eq) was reacted at 60 ℃ for 3 hours. The solution was cooled to 23 ℃ and added dropwise to ice water (800mL) in portions over 15 minutes with stirring. After the addition was complete, the mixture was stirred for an additional 15 minutes and diluted with dichloromethane (500 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2X 200 mL). The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo to give intermediate-1A (12.5g, 103% yield) as a tan solid.

¹H NMR(500MHz,DMSO-d₆)δ9.11(d,1H),9.04(s,1H),7.71–7.68(m,1H),7.37–7.30(m,2H),7.25–7.20(m,1H),7.12(t,1H),6.92(td,1H),5.95(s,2H)。

Synthesis of intermediate-9

Except that 5,6,7, 8-tetrahydro- [1,2,4 ]]Triazolo [4,3-a]Pyrazine-3-carboxylic acid ethyl ester (4 equivalents) was prepared as the target compound from intermediate-1A in accordance with general procedure B, in addition to the amine reactant, and the reaction was carried out in THF. Work-up was carried out with dichloromethane and brine. The crude product was purified by silica gel chromatography eluting with a 0-10% methanol/dichloromethane mobile phase gradient to give the desired solid intermediate-9 (42mg, 37% yield).¹H-NMR(400MHz,CDCl3)δ8.47(d,1H),8.35(d,1H),7.40(s,1H),7.21-7.16(m,1H),7.01(t,1H),6.95(t,1H),6.84(t,1H),6.65(d,1H),5.98(s,2H),5.35(s,2H),4.59(t,2H),4.48(q,2H),4.30(t,2H),1.44(t,3H)。

Synthesis of intermediate-8

The title compound was prepared from intermediate-1A following general procedure B, except that 3-amino-2, 2-difluoropropionic acid was the amine reactant. The reaction was heated in a solution of dioxane/water (10:1) at 110 ℃ for 18 h. The reaction was concentrated in vacuo, methanol was added and the crude product was purified by reverse phase HPLC to give the desired intermediate-8 (20mg, 22% yield).¹H NMR(500MHz,CD₃OD)δppm 8.78(d,1H),8.22(d,1H),7.61(s,1H),7.25-7.31(m,1H),7.07-7.12(m,1H),7.05(t,1H),6.96(d,1H),6.89(t,1H),6.00(s,2H),4.35(t,2H).

Synthesis of intermediate-13

2- (1- (2-Fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5-nitropyrimidin-4-ol (the starting material is described in the previously published patent application WO2012/3405A 1) (25mg, 1 eq.) with POCl₃(457. mu.l, 75 equivalents) and stirred under reflux for 1.5 h. The reaction was concentrated in vacuo and the residue azeotroped with toluene (x 2). The residue was redissolved in THF (0.7mL) and morpholine (171. mu.l, 30 equivalents) was added. The reaction was heated to 40 ℃ and stirred at this temperature for 1.5 hours. The residue was transferred to a 1:1 mixture of ethyl acetate and water. The layers were separated and the aqueous layer was extracted 3 times with ethyl acetate. The organic fractions were combined and washed with brine. The organic layer was washed with MgSO ₄Drying, filtration and concentration in vacuo gave the desired compound (30mg, 97%) as a pale yellow solid.

¹H-NMR(400MHz,CDCl₃)δ8.47(d,1H),8.36(d,1H),8.09-8.16(m,1H),7.69(dd,1H),7.41(d,1H),7.20(t,1H),6.66-6.70(m,1H),6.45(d,1H),6.06(s,2H),3.79-3.86(m,4H),3.74(m,4H)。

Synthesis of intermediate-3

The target compound was synthesized by 3 steps:

step 1: synthesis of 2- (trifluoromethyl) oxirane-2-carboxamide

To a solution of 2- (bromomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide (1 eq) in acetone was added potassium carbonate (2 eq). The mixture was heated to reflux for 2 hours. The mixture was concentrated in vacuo. The resulting residue was diluted with water and extracted with ethyl acetate. The organic layer was dried, filtered and evaporated to give 2- (trifluoromethyl) oxirane-2-carboxamide (1.44g, 76% yield) as a yellow gum.

¹H NMR(500MHz,CD₃OD)δppm 3.17(dd,2H)。

Step 2: synthesis of 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide

A mixture of ammonia [7M in methanol ] (10 equivalents) and 2- (trifluoromethyl) oxirane-2-carboxamide (1 equivalent) was stirred in a sealed vial at 80 ℃ for 24 hours. The mixture was concentrated in vacuo to give 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide (1.3g, 84% yield) as a brown gum.

¹H NMR(500MHz,DMSO-d₆)δ3.01-3.11(m,1H),2.84(d,1H)。

Step 3: synthesis of intermediate-3

The title compound was prepared from intermediate-1A following general procedure B, except that 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide (4 equivalents) was the amine reactant. A dioxane/water (3:1) solution of the reactants was heated at 90 ℃ for 24 hours using 4 equivalents of triethylamine. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give a solid. The solid was purified by silica gel chromatography (gradient 0 to 80% ethyl acetate in hexanes) to give the desired intermediate-3 (262mg, 40% yield) as a white solid.

¹H NMR(500MHz,DMSO-d₆)δppm 9.08-9.13(m,1H),8.33(d,1H),7.49-7.55(m,1H),7.28-7.37(m,1H),7.17-7.25(m,2H),7.10(t,1H),6.98(t,1H),5.86-5.92(m,2H),3.92-4.04(m,2H)。

Synthesis of intermediate-5D

The target compound was synthesized by 3 steps:

step 1: synthesis of ethyl 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxylate

To a solution of ethyl 3-methyl-1H-pyrazole-5-carboxylate in DMF was added sodium hydride (60 wt% in mineral oil, 1.2 equivalents). After 10 minutes, 2-fluorobenzyl bromide (1.2 eq) was added and the reaction stirred for 20 hours. Water was added to the reaction solution, and the resulting mixture was extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried over sodium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (gradient 10-40% ethyl acetate/hexanes) gave ethyl 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxylate (79% yield) and ethyl 1- (2-fluorobenzyl) -3-methyl-1H-pyrazole-5-carboxylate (9% yield).

Step 2: synthesis of 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxylic acid

To a solution of ethyl 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxylate in THF/MeOH/water (3:1:1 ratio) was added lithium hydroxide hydrate (1.5 equiv). After 23 hours of reaction, the volatile organics were removed in vacuo and the resulting mixture was acidified with 1N HCl to pH 3. Filtration in vacuo afforded 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxylic acid (92% yield).

Step 3: synthesis of 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carbonitrile

To a suspension of 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxylic acid, 2-methylpropan-2-amine (3 eq) and triethylamine (2 eq) in ethyl acetate was added n-propylphosphonic anhydride (T3P, 50 wt% in ethyl acetate, 3 eq). The resulting yellow solution was heated at 65 ℃ for 2.5 hours. The solvent was removed in vacuo. Phosphorus trichloride (12 equivalents) was added and the resulting mixture was stirred at 70 ℃ for 1 hour 40 minutes. The reaction was quenched by carefully pouring into a mixture of water and ice, neutralized to pH 7 by addition of saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed in vacuo. Purification by silica gel chromatography (gradient 10% ethyl acetate/hexanes) gave 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carbonitrile (49% yield).

Step 4: synthesis of 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxamidine

To a solution of 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carbonitrile in methanol was added sodium methoxide (25 wt% MeOH solution, 5 eq) and stirred for 24H. Ammonium chloride (10 equivalents) was added. After 26 hours, the reaction mixture was concentrated in vacuo and diluted with half-saturated sodium bicarbonate and ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. Starting materials remained due to incomplete reaction. The mixture was again treated under the above conditions to give 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxamidine (92% yield).

Step 5: synthesis of intermediate-5D

Sodium (Z) -3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (see general procedure a, step 4, 3.0 equiv.) was added to a suspension of 1- (2-fluorobenzyl) -5-methyl-1H-pyrazole-3-carboxamidine and heated at 90 ℃ for 1H. After cooling to room temperature, the reaction mixture was neutralized by adding HCl (1.25M in EtOH). The resulting brown suspension was concentrated in vacuo. The residue was diluted with dichloromethane and water, and the aqueous layer was extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed in vacuo. Slurried with dichloromethane to give the title compound (206mg, 62% yield) as a white solid.

¹H-NMR(500MHz,DMSO-d₆)δ12.9(br s,1H),8.07(br s,1H),7.38(app.q,1H),7.25(m,1H),7.18(app.t,1H),7.11(m,1H),6.72(s,1H),5.44(s,2H),2.30(s,3H)。

Synthesis of intermediate-12

The target compound was prepared by 2 steps:

step 1: synthesis of diethyl 2- (dicyanomethyl) -2-methylmalonate

A solution of diethyl 2-bromo-2-methylmalonate (1 eq), malononitrile (1 eq) and potassium tert-butoxide (1 eq) in THF was heated to reflux for 15 hours. The mixture was diluted with ethyl acetate and saturated aqueous ammonium chloride solution, and the phases were separated. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give an oil. The oil was purified by silica gel chromatography (gradient 10-15% ethyl acetate in hexanes) to give diethyl 2- (dicyanomethyl) -2-methylmalonate (5.76g, 32% yield) as a colorless oil.

¹H NMR(500MHz,CDCl₃)δppm 4.53(s,1H),4.27-4.39(m,4H),1.81(s,3H),1.33(t,6H)。

Step 2: synthesis of intermediate-12

1- (2-Fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine hydrochloride (prepared by step 3 of general procedure A by using 1- (isoxazol-3-yl) ethanone in step 1 and 2-fluorobenzylhydrazine in step 2) (1 eq), diethyl 2- (dicyanomethyl) -2-methylmalonate (equiv) and potassium bicarbonate (2 eq) in t-BuOH solution was heated at reflux for 5H. After cooling, water was added to the reaction mixture and stirred for 30 minutes. The precipitate was filtered, washed with a minimum amount of water and diethyl ether, dried under high vacuum overnight,intermediate-12 (385mg, 52% yield) was obtained as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 11.30(s,1H),9.10(d,1H),7.38(s,1H),7.29-7.36(m,1H),7.18-7.26(m,2H),7.08-7.14(m,1H),6.81-6.90(m,1H),6.65(br.s.,2H),5.88(s,2H),4.04-4.16(m,2H),1.59(s,3H),1.11(t,3H).

Synthesis of intermediate-11

Ammonia (7.0M in MeOH) (200 eq) was added to intermediate-12 (1 eq). The reaction mixture was heated at 50 ℃ for 16 hours. The resulting solution was then concentrated in vacuo and the residue was purified by reverse phase HPLC (5-60% aqueous acetonitrile containing 1% TFA) to give intermediate-11 as a white solid (24mg, 63% yield).¹H NMR(400MHz,DMSO-d₆)δppm11.35(br.s.,1H),9.08-9.13(m,1H),7.47(s,1H),7.43(s,1H),7.28-7.38(m,1H),7.23-7.27(m,1H),7.17-7.23(m,2H),7.06-7.14(m,1H),6.77-7.00(m,3H),5.91(s,2H),1.56(s,3H)。

Synthesis of intermediate-5B

A suspension of intermediate-5A and sodium methoxide in methanol (0.5M solution, 4 equivalents) was heated in a microwave container at 130 ℃ for 4 hours. The reaction was quenched with 1N HCl solution to pH 2 and the resulting residue was filtered. The solid was washed with methanol and dried in vacuo to give the desired compound (1.45g, 68%) as a white solid. ¹H NMR(500MHz,CD₃OD)δppm 8.04(d,1H),7.71(s,1H),7.23-7.36(m,1H),7.00-7.18(m,2H),6.90(t,1H),5.94(s,2H),2.56(s,3H).

Synthesis of intermediate-1B

Phosphorus oxychloride (60 equivalents) was added to intermediate-5B and the resulting mixture was stirred at 45 ℃ until the reaction was complete as judged by LC/MS. The reaction was then carefully poured onto ice, extracted with 4:1 dichloromethane/isopropanol and the layers separated. The organic phases were combined, dried over sodium sulfate, filtered and concentrated in vacuo. This material was used in the next step without further purification.

Synthesis of intermediate-6

Following general procedure B, the title compound was prepared using 1- ((methylamino) methyl) cyclopropanecarboxylic acid as the amine reactant, intermediate-1B was used in place of intermediate-1A, and a dioxane solution of the reactant was heated at 100 ℃ for 36 hours. The crude product was purified by reverse phase HPLC to give the desired intermediate-6 (50mg, 69% yield) as a brown solid.¹H NMR(500MHz,DMSO-d6)δppm 12.53(br.s,1H),8.19(d,1H),7.65(s,1H),7.33(d,1H),7.17-7.26(m,1H),7.11(t,1H),6.86(t,1H),5.81(s,2H),4.00(s,2H),3.24(d,3H),2.57(s,3H),1.03(d,2H),0.74-0.91(m,2H)。

Synthesis of intermediate-4

The target compound was synthesized by 3 steps:

step 1: synthesis of 2- (bromomethyl) -3,3, 3-trifluoro-2-hydroxypropionic acid

A mixture of 2- (bromomethyl) -3,3, 3-trifluoro-2-hydroxypropionitrile (1 eq), water (1 eq) and concentrated sulfuric acid (4 eq) was heated in a sealed vial to 110 ℃ for 1 hour. The mixture was poured onto ice and extracted with ether. The organic layer was washed with MgSO₄Drying, filtration and concentration in vacuo gave 2- (bromomethyl) -3,3, 3-trifluoro-2-hydroxypropionic acid (1.3g, 33% yield) as a clear oil.

¹H NMR(500MHz,CDCl₃)δppm 3.89(d,1H),3.63-3.69(m,1H)。

Step 2: synthesis of 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionic acid

A mixture of ammonium hydroxide [ 28% aqueous solution ] (10 equivalents) and 2- (bromomethyl) -3,3, 3-trifluoro-2-hydroxypropionic acid (1 equivalent) was stirred at 23 ℃ for 24 hours. The mixture was concentrated in vacuo. The resulting solid was treated with a minimum amount of ethanol. The precipitate was collected by filtration and dried under vacuum to give 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionic acid (412mg, yield 43%) as a white solid.

¹H NMR(500MHz,DMSO-d₆)δppm 2.86-3.27(m,2H)。

Step 3: synthesis of intermediate-4

The title compound was prepared according to general procedure B, except 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionic acid (4 equivalents) was the amine reactant. 6 equivalents of triethylamine were used and a 1, 4-dioxane/water (4:1) solution of the reactants was heated to 85 ℃ for 24 hours. The mixture was cooled to 23 ℃ and diluted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution and MgSO₄Drying, filtering and vacuum concentrating to obtain a crude product. The crude product was purified by silica gel chromatography eluting with a gradient of 0-100% ethyl acetate in hexanes to give intermediate-4 as a white solid (50mg, 7% yield from step 3).¹H NMR(500MHz,DMSO-d₆)δppm 8.28(d,1H),7.59(t,1H),7.46(s,1H),7.30-7.36(m,1H),7.16-7.24(m,2H),7.10(t,1H),6.91(t,1H),5.88(s,2H),4.24(dd,1H),3.84(dd,1H)。

Synthesis of intermediate-7

1- (2-Fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine hydrochloride (prepared by general method A, step 3, 1 eq), tert-butyl 4-oxotetrahydrothiophene-3-carboxylate (3 eq) and 1, 8-diazabicyclo [5.4.0 ]A solution of undec-7-ene (1 eq) in pyridine was heated to 80 ℃ for 12 hours. The reaction was concentrated in vacuo, slurried with methanol, concentrated in vacuo, and slurried again with methanol. The precipitate was filtered and dried to give the desired episulfide intermediate (190mg, yield 45%) as a pale brown solid. To a solution of the sulfide intermediate (1 eq) in dichloromethane was added peracetic acid (2.3 eq). After 30 minutes of reaction, the reaction was concentrated in vacuo, slurried with water, and filtered to give the desired intermediate-7 (148.8mg, 73% yield) as an off-white solid.¹H-NMR(500MHz,CDCl₃)δ10.2(br.s,1H),8.56(s,1H),7.31–7.34(m,1H),7.30(s,1H),7.07–7.12(m,3H),6.64(m,1H),5.93(s,2H),4.36(s,2H),4.35(s,2H)。

Synthesis of intermediate-10

The target compound was synthesized by 2 steps:

step 1：

Following general procedure B, intermediate-13 was used in place of intermediate-1A, 2- (aminomethyl) -1,1,1,3,3, 3-hexafluoropropan-2-ol (1.5 equivalents) as the amine reactant, 3 equivalents of triethylamine, the reactants dioxane: the water (3:1) solution was heated at 30 ℃ for 1 hour. The reaction was cooled and diluted with ethyl acetate. The organic layer was washed with water (2 times) and brine, then dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography using a gradient of 0-100% ethyl acetate/hexanes to give the desired intermediate (77mg, 73% yield) as a white solid. ¹H-NMR(400MHz,CDCl₃)δppm 9.36(s,1H),8.59(m,1H),8.55(d,1H),7.64(br s,1H),7.42(s,1H),7.28(m,1H),7.08(m,1H),7.06(m,1H),6.64(d,1H),5.98(s,2H),4.27,(d,2H)。

Step 2: synthesis of intermediate-10

The methanol solution of the intermediate (1 eq) obtained in step 1 was replaced by hydrogen by the addition of 10% palladium on carbon (0.2 eq) at 23 ℃. Subjecting the mixture to reaction under positive H₂Stirred under pressure for 1 hour and filtered through celite. The filter cake was washed with methanol and the combined washings were concentrated in vacuo. The resulting crude residue was purified by silica gel chromatography using a gradient of ethyl acetate in hexane to afford the desired intermediate-10 (53mg, 66% yield) as a white solid.¹H-NMR(400MHz,CDCl₃)δppm 9.39(s,1H),7.92(br s,1H),7.19(m,1H),7.13(m,2H),7.98(m,1H),6.92(m,2H),6.52(s,1H),5.85(s,2H),4.01,(s,2H)。

Synthesis of intermediate-5C

The target compound was synthesized by 4 steps:

step 1: synthesis of (3,3, 3-trifluoropropyl) hydrazine hydrochloride

3-bromo-1, 1, 1-trifluoropropane (1 eq) and hydrazine hydrate (10 eq) were dissolved in absolute ethanol and heated at 80 ℃ for 18 hours. The solution was cooled to 23 ℃ and concentrated in vacuo at 15 ℃. The oily crude product was diluted with water and dichloromethane, and solid potassium carbonate was then added to saturate the aqueous layer. The phases were mixed and separated, then the aqueous phase was extracted 2 times with additional dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo to give a colorless oil. A small portion of the neutral hydrazine product was removed for characterization by NMR. The residue was dissolved in ether and treated with hydrochloric acid (2.5M ethanol solution) and concentrated in vacuo to give the desired intermediate (3,3, 3-trifluoropropyl) hydrazine hydrochloride (2.02g, 43% yield) as a white solid. ¹H-NMR(400MHz,CDCl₃)δppm 3.18(br s,4H),3.02(m,2H),2.36(m,2H)。

Step 2: synthesis of ethyl 3- (isoxazol-3-yl) -1- (3,3, 3-trifluoropropyl) -1H-pyrazole-5-carboxylate

A solution of (3,3, 3-trifluoropropyl) hydrazine hydrochloride (1 eq) in ethanol/water (9:1) was added potassium carbonate (0.6 eq) at 23 ℃ followed by ethyl 4- (isoxazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoate (2 eq, generated by using 1- (isoxazol-3-yl) ethanone in step 1, via step 1 of general procedure A). The solution was stirred at 23 ℃ for 2 days, and then 6N hydrochloric acid (1.5 equivalents) was added dropwise to the reaction. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate. The organics were washed 5 times with water, washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with an ethyl acetate/dichloromethane gradient to give the desired pyrazole ester, ethyl 3- (isoxazol-3-yl) -1- (3,3, 3-trifluoropropyl) -1H-pyrazole-5-carboxylate (1.34g, 36% yield) as a pale yellow solid.¹H-NMR(400MHz,CDCl₃)δppm 8.55(d,1H),7.15(s,1H),6.63(d,1H),4.95(m,2H),4.46(q,2H),2.85(m,2H),1.44(t,3H)。

Step 3: synthesis of 5- (isoxazol-3-yl) -1- (3,3, 3-trifluoropropyl) -1H-pyrazole-3-carboxamidine

The desired amidine intermediate was prepared as described in step 3 of general procedure A using 3- (isoxazol-3-yl) -1- (3,3, 3-trifluoropropyl) -1H-pyrazole-5-carboxylic acid ethyl ester as starting ester. The reaction was heated at 110 ℃ for 4 hours. The reaction mixture was cooled in ice and then methanol (14 eq) and aqueous hydrochloric acid (17 eq) were added continuously over 5 minutes. The mixture was heated at 80 ℃ for 30 minutes, then cooled in ice and filtered. The filter cake was washed 2 times with toluene and air dried to give the crude amidine hydrochloride. The material was stirred in saturated aqueous sodium carbonate solution and extracted with ethyl acetate/isopropanol (5:1 mixture). The organic phase is washed with water and brine, dried over sodium sulfate, filtered and Concentration in vacuo afforded the desired neutral amidine, 5- (isoxazol-3-yl) -1- (3,3, 3-trifluoropropyl) -1H-pyrazole-3-carboxamidine, as a pale yellow solid.¹H-NMR(400MHz,CDCl₃)δppm 8.45(d,1H),6.99(s,1H),6.55(d,1H),5.61(br.s.,3H),4.83–4.74(m,2H),2.81–2.65(m,2H)。

Step 4: synthesis of intermediate-5C

The title product was prepared according to general procedure A, step 4, using 5- (isoxazol-3-yl) -1- (3,3, 3-trifluoropropyl) -1H-pyrazole-3-carboxamidine as starting amidine. 2.5 equivalents of sodium (Z) -3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol were used and the reaction was heated at 90 ℃ for 2 hours. The reaction was cooled to 23 ℃ and the solvent was removed in vacuo. The residue was redissolved in dichloromethane and treated with hydrochloric acid (2.5M in ethanol, 3 equivalents). The resulting solid was filtered, washed 2 times with dichloromethane, and air-dried to give the desired compound (0.43g, yield 110%) as a white solid.¹H-NMR(400MHz,CD₃OD)δppm 8.84(d,1H),8.03(d,1H),7.40(s,1H),6.95(d,1H),4.96(t,2H),2.92(m,2H)。

Synthesis of intermediate-16

The target compound was prepared by 2 steps:

step 1: (E) synthesis of (E) -2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5- (phenyldiazenyl) pyrimidine-4, 6-diamine

A solution of 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine (prepared by the method of step 3 of general procedure A to prepare intermediate 1A, 1 eq), (E) -2- (phenyldiazenyl) malononitrile (1.2 eq) and potassium bicarbonate (2 eq) in t-BuOH was heated at reflux for 18H. After cooling, the reaction mixture was concentrated in vacuo and used in the next step without further purification.

Step 2: synthesis of intermediate-16

A solution of (E) -2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5- (phenyldiazenyl) pyrimidine-4, 6-diamine (1 eq) and 20% palladium on carbon (0.5 eq) in DMF was stirred with hydrogen at 23 ℃ for 18H. The reaction mixture was then filtered through celite and the residue was washed with DMF and then with a small amount of methanol. The filtrate was concentrated in vacuo, the residue suspended in ethyl acetate containing one drop of methanol and stirred vigorously. The precipitate was filtered, washed with ethyl acetate and dried in vacuo to give the desired triaminopyrimidine intermediate, 2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -pyrimidine-4, 5, 6-triamine (278mg, 46% in 2 steps) as a dark yellow solid.

Synthesis of intermediate-14

A solution of piperidine-4-carboxylic acid (3 equiv.), triethylamine (10 equiv.), and intermediate-1A in tetrahydrofuran and water (1:1 ratio) was stirred at 100 deg.C until complete consumption of the starting material was judged by LC/MS, and the solution was diluted with 1N aqueous hydrochloric acid and ethyl acetate as per general procedure B. The layers were separated and the aqueous layer was extracted with ethyl acetate and 5:1 dichloromethane/isopropanol. The organics were combined, dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by reverse phase HPLC (5-75% aqueous acetonitrile containing 0.1% trifluoroacetic acid, 20 min gradient) gave intermediate-14 (11mg, 44% yield) as a white solid. ¹H-NMR(400MHz,CD₃OD)δ8.79(m,1H),8.23(d,1H),7.57(m,1H),7.31-7.26(m,1H),7.12-7.03(m,2H),6.96(m,1H),6.90(t,1H),5.99(s,2H),4.70(d,2H),3.51-3.45(m,2H),2.79-2.74(m,1H),2.15-2.11(m,2H),1.90-1.80(m,2H)。

Compound 25

A solution of intermediate-3 (1 eq) and NBS (1.2 eq) in DMF was stirred at 23 ℃ for 24 h. The reaction was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give the crude product as an oil. The oil was purified by column chromatography to give the desired compound (27mg, yield 4%) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.24(d,1H),8.37(d,1H),7.86(br.s.,1H),7.31-7.37(m,1H),7.09-7.20(m,3H),5.68(s,2H),4.04(d,2H)。

Compound 52

The target compound was synthesized by 3 steps:

step 1: synthesis of 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionylhydrazine

A solution of (diazomethyl) trimethylsilane (2 equivalents) and intermediate-4 (1 equivalent) in THF (3.0ml) was heated at 80 deg.C for 4 hours. The mixture was cooled to 23 ℃ and concentrated in vacuo to afford the desired intermediate ester. Intermediate (1 eq) was combined with water (11 eq), anhydrous hydrazine (130 eq) and methanol and heated to 50 ℃ for 2 hours. After the reaction was complete, the excess hydrazine was removed using methanol and benzene as azeotropes. The resulting residue was further dried in vacuo to give the desired intermediate, 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (257mg, 64% yield) as a pale yellow solid. ¹H NMR(500MHz,DMSO-d₆)δppm 9.11(d,1H),8.33(d,1H),7.52(s,1H),7.27-7.41(m,1H),7.18-7.26(m,2H),7.11(t,1H),6.96(t,1H),5.90(s,2H),3.98(br.s.,2H).

Step 2: synthesis of N' -acetyl-3, 3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl)) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide

To a solution of potassium carbonate (5 equiv) and 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (1 equiv) in THF/water (1:1) was added acetyl chloride (1.5 equiv). The mixture was stirred at 23 ℃ for 1 hour. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered, and concentrated in vacuo to give the crude product as a solid. The solid was purified by silica gel chromatography to give the desired intermediate, N' -acetyl-3, 3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl)) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (190mg, 64% yield) as a light yellow solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.08-9.12(m,1H),8.06(s,1H),7.50-7.56(m,1H),7.32(d,1H),7.17-7.26(m,2H),7.10(t,1H),6.89-6.99(m,1H),5.84-5.96(m,2H),3.91-4.17(m,2H),1.85(s,3H).

Step 3: synthesis of Compound 52

To a 0 ℃ cooled solution of N' -acetyl-3, 3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl)) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (1 eq) in pyridine was added trifluoromethanesulfonic anhydride (5 eq). The mixture was removed from the ice bath and stirred at 23 ℃ for 24 hours. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give the crude product as an oil which was purified by silica gel chromatography. The material was further washed with a minimum amount of methanol and dichloromethane, collected by filtration and dried in vacuo to give the desired compound (48mg, 26% yield) as a brown solid. ¹H NMR(500MHz,DMSO-d₆)δppm 9.11(d,1H),8.26(d,1H),7.40(s,1H),7.31-7.37(m,1H),7.22(d,1H),7.19(d,1H),7.12(t,1H),6.94(t,1H),5.84-5.92(m,2H),4.29(dd,1H),4.17(dd,1H),2.25(s,3H)。

Compound 69

The target compound is synthesized through 2 steps:

step 1: synthesis of N' - (3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionyl) cyclopropanecarboxylic acid hydrazide

To a 1:1 mixed solution of THF and water containing potassium bicarbonate (5 equivalents) and 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (as described for compound 52, in the synthesis of step 1) (1 equivalent) was added cyclopropanecarbonyl chloride (5 equivalents). The mixture was stirred at 23 ℃ for 1 hour. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give the desired intermediate, N' - (3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionyl) cyclopropanecarbohydrazide (206mg, 51% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.10(d,1H),8.32(d,1H),7.52(s,1H),7.33(s,1H),7.16-7.26(m,2H),7.09(t,1H),6.93(s,1H),5.90(s,2H),4.07-4.14(m,2H),1.59(d,1H),0.66-0.76(m,4H)。

Step 2: synthesis of 2- (5-cyclopropyl-1, 3, 4-oxadiazol-2-yl) -1,1, 1-trifluoro-3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propan-2-ol

To a cold solution of N' - (3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionyl) cyclopropanecarbohydrazide (1 eq) in pyridine was added trifluoromethanesulfonic anhydride (4 eq). Inverse direction After completion, the mixture was diluted with ethyl acetate and washed with 1N HCl solution. The organic layer was dried, filtered and evaporated to give the crude product as an oil. The oil was purified by silica gel chromatography to give the desired compound (9mg, 9% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.08-9.13(m,1H),8.27(d,1H),7.37(s,1H),7.28-7.36(m,1H),7.19-7.25(m,1H),7.17(d,1H),7.11(t,1H),6.93(t,1H),5.87(s,2H),4.08-4.16(m,2H),1.97-2.08(m,1H),0.93(dd,2H),0.74(dd,2H)。

Compound 61

The target compound is synthesized through 3 steps:

step 1: synthesis of 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylic acid hydrazide

To a suspension of 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine hydrochloride (prepared by the method described in step 3 of general procedure A using 1- (isoxazol-3-yl) ethanone in step 1 and 2-fluorobenzylhydrazine in step 2, 1 eq) in ethanol was added triethylamine (4 eq). To this mixture was added hydrazine monohydrate (1 eq). The mixture was stirred at 23 ℃ for 24 hours and concentrated in vacuo. The resulting residue was diluted with ethyl acetate and washed with brine. The organic layer was dried, filtered and evaporated to give the desired intermediate, 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylic acid hydrazide (461mg, 99% yield) as a pale yellow solid.¹H NMR(500MHz,CD₃OD)δppm 8.75(d,1H),7.18-7.40(m,1H),6.97-7.15(m,3H),6.79-6.92(m,2H),5.82-5.97(m,2H)。

Step 2: synthesis of ethyl 2- (3- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5-hydroxy-1, 2, 4-triazin-6-yl) -2-methylpropionate (Compound 110)

An ethanol solution containing 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboximide (1 eq), potassium bicarbonate (1.2 eq) and diethyl 2, 2-dimethyl-3-oxosuccinate (1.2 eq) was heated at 80 ℃ for 24H and concentrated in vacuo. The resulting residue was diluted with ethyl acetate and washed with brine. The organic layer was dried, filtered and evaporated to give an oil. Purification by column chromatography gave the desired intermediate, ethyl 2- (3- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5-hydroxy-1, 2, 4-triazin-6-yl) -2-methylpropionate (400mg, 34% yield) as a pale yellow solid.¹H NMR(500MHz,CD₃OD)δppm 8.80(d,1H),8.77(d,1H),7.53(s,1H),7.36(s,1H),7.26-7.33(m,3H),6.03(s,2H),4.11-4.17(m,2H),1.53(s,6H),1.22-1.27(m,3H)。

Step 3: synthesis of Compound 61

A mixture containing ethyl 2- (3- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5-hydroxy-1, 2, 4-triazin-6-yl) -2-methylpropionate (compound 110) and phosphorus oxychloride (10 eq) was stirred at 23 ℃ for 2 hours. The mixture was concentrated in vacuo. To this mixture was added 7N ammonia in methanol (4 equivalents) and additional methanol. The reaction was stirred at 23 ℃ for 30 minutes. Filtration afforded the desired compound (8.3mg, 8% yield) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.78-8.80(m,1H),7.64(s,1H),7.26-7.32(m,1H),7.08-7.14(m,1H),7.05(t,1H),6.95-6.98(m,1H),6.87(t,1H),6.00-6.05(m,2H),4.17(s,3H),4.15(q,2H),1.66(s,6H),1.15-1.21(m,3H)。

Compound 70

A1, 4-dioxane/water (3:1) solution containing musk quinone (1.5 equivalents), trimethylamine (1.5 equivalents) and intermediate-1B (1 equivalent) was heated to 70 ℃ for 24 hours. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give the crude product as an oil. The oil was purified by silica gel chromatography (0 to 100% ethyl acetate in hexanes) to give the desired compound (13mg, 21% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.26(d,1H),7.68(s,1H),7.29-7.36(m,1H),7.18-7.26(m,2H),7.11(t,1H),6.85(t,1H),5.82(s,2H),3.31(s,2H),2.57(d,3H)。

Compound 71

A solution of 2- (trifluoromethyl) piperazine (3 eq), triethylamine (3 eq) and intermediate-1B (1 eq) in 1, 4-dioxane/water (3:1) was heated to 80 ℃ for 1 hour. The mixture was diluted with ethyl acetate. The organic layer was washed with water, dried, filtered and evaporated to give a crude oil. The oil was purified by silica gel chromatography (0 to 100% ethyl acetate in hexanes) to give the desired compound (40mg, 60% yield) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.24(d,1H),7.66(s,1H),7.20-7.39(m,1H),7.03-7.15(m,2H),6.83(t,1H),5.84(s,2H),4.67(d,1H),4.42(d,1H),3.58(t,1H),3.37-3.48(m,2H),3.15(d,1H),2.87-3.02(m,1H),2.58(s,3H)。

Compound 72

An aqueous solution of 1, 4-dioxane/water containing (S) -2- (aminomethyl) -3-methylbutyric acid hydrochloride (4 equivalents), triethylamine (2 equivalents) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) was heated to 70 ℃ for 24 hours. The mixture was diluted with ethyl acetate. The organic layer was washed with 1N HCl, dried, filtered and evaporated to give a solid. The solid was purified by silica gel chromatography (eluting with 0 to 10% methanol in dichloromethane) to give the desired compound (14mg, 29% yield) as white A colored solid.¹H NMR(500MHz,CD₃OD)δppm 8.04(d,1H),7.63-7.69(m,1H),7.27(q,1H),7.00-7.15(m,2H),6.83(t,1H),5.86-5.95(m,2H),3.70-3.93(m,2H),2.63-2.74(m,1H),2.56(s,3H),1.93-2.07(m,1H),1.02-1.15(m,6H)。

Compound 73 and compound 74

The target compound is synthesized through 3 steps:

step 1：

Synthesis of (S) -2- (((tert-butoxycarbonyl) amino) methyl) -3-methylbutyric acid

A methanol solution containing di-tert-butyl dicarbonate (2 equivalents), triethylamine (1 equivalent) and (S) -2- (aminomethyl) -3-methylbutyric acid (1 equivalent) was stirred at 23 ℃ for 24 hours. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate and washed with 1N HCl solution. The organic layer was dried, filtered and evaporated to give the desired intermediate, (S) -2- (((tert-butoxycarbonyl) -amino) methyl) -3-methylbutyric acid (730mg, 100% yield) as a white solid.¹H NMR(500MHz,CDCl₃)δppm 3.39-3.55(m,1H)3.06-3.31(m,1H)2.40-2.58(m,1H)1.86-2.10(m,1H)1.38-1.52(m,9H)0.94-1.05(m,6H)。

Step 2：

Synthesis of (S) -2- (((tert-butoxycarbonyl) amino) methyl) -3-methylbutyric acid

To a solution of (S) -2- (((tert-butoxycarbonyl) amino) methyl) -3-methylbutyric acid (1 eq) in THF at 0 deg.C was added sodium hydride [ 60% dispersion in mineral oil](10 equivalents). The mixture was stirred at 0 ℃ for 15 minutes. To this mixture was added methyl iodide (10 equivalents). The mixture was removed from the ice bath and stirred at 23 ℃ for 24 hours. The mixture was diluted with ethyl acetate and washed with 1N HCl solution. The organic layer was dried, filtered and evaporatedThen, an oil was obtained. The oil was purified by column chromatography (0 to 30% ethyl acetate in hexanes) to afford the desired intermediate, (S) -2- (((tert-butoxycarbonyl) (methyl) amino) methyl) -3-methylbutyric acid (186mg, 25% yield) as a yellow oil. ¹H NMR(500MHz,CDCl₃)δppm 3.50-3.61(m,1H)3.29-3.41(m,1H)2.87(s,3H)2.47-2.68(m,1H)1.91(d,1H)1.46(s,9H)0.96-1.06(m,6H)。

Step 3: preparation of Compound 73 and Compound 74

A solution of 1.25M HCl (10 equivalents) and (S) -2- (((tert-butoxycarbonyl) (methyl) amino) methyl) -3-methylbutyric acid (1 equivalent) in ethanol was stirred at 23 ℃ for 24 h. The mixture was concentrated in vacuo. To this mixture was added triethylamine (3 equivalents), 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent), 1, 4-dioxane and water (3: 1). The mixture was heated to 40 ℃ for 4 hours. The mixture was diluted with ethyl acetate and washed with 1N HCl solution. The organic layer was dried, filtered and evaporated to give an oil. Purification of the oil by column chromatography (0 to 100% ethyl acetate in hexanes elution) gave two products, compound 73 as a white solid (5mg, 4% yield) and compound 74 as a pale yellow oil (44mg, 36% yield).

Compound 73:¹H NMR(500MHz,CD₃OD)δppm 8.12(d,1H)7.65(s,1H),7.24-7.32(m,1H),7.10(s,1H),7.04(t,1H),6.82(t,1H),5.85-5.99(m,2H),4.17(dd,1H),3.84(dd,1H),3.36(d,3H),2.66-2.74(m,1H),2.55(s,3H),1.90-1.99(m,1H),0.99-1.15(m,6H)。

compound 74:¹H NMR(500MHz,CD₃OD)δppm 8.12(d,1H),7.64(s,1H),7.23-7.32(m,1H),7.08-7.14(m,1H),7.04(t,1H),6.83(t,1H),5.90(s,2H),3.99-4.10(m,3H),3.91(dd,1H),3.31(d,3H),2.68-2.77(m,1H),2.56(s,3H),1.90-2.03(m,1H),1.07-1.16(m,6H),0.99(d,3H)。

compound 4

To a suspension of intermediate-2 (1 eq) in dichloromethane was added a solution of oxalyl chloride (3 eq) in 2M dichloromethane, followed by triethylamine (3 eq) at 23 ℃. After 10 minutes, the reaction mixture was concentrated in vacuo, diluted with tetrahydrofuran and treated with saturated ammonium hydroxide solution. The reaction mixture became golden, and then the reaction was acidified with a 1N hydrochloric acid solution and extracted with ethyl acetate. The combined organic layers were washed with 1N hydrochloric acid solution, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 5-95% acetonitrile in water to afford the desired compound, compound 4(2.3mg, 4% yield) as a white solid. ¹H-NMR(500MHz,DMSO-d₆)δppm 10.19(s,1H),9.07(d,1H),8.82(d,1H),8.52(s,1H),8.17(s,1H),7.95(d,1H),7.69(s,1H),7.28–7.32(m,1H),7.24(d,1H),7.18–7.22(m,1H),7.07–7.10(m,1H),6.85–6.88(m,1H),5.91(s,2H)。

Compound 17

To a solution of intermediate-2 (1 eq) in dichloromethane was added methyl 3-chloro-3-oxopropanoate (1.15 eq) at room temperature followed by triethylamine (1.5 eq). After stirring at 23 ℃ for 1 hour, the reaction mixture was poured into a 1N hydrochloric acid solution, extracted with dichloromethane and then with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to afford a solid. The crude product was purified by silica gel chromatography eluting with a gradient of 1-8% methanol in dichloromethane to give the desired intermediate, methyl 3- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3-oxopropanoate (173mg, 65% yield) as an off-white solid.

To methyl 3- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3-oxopropanoate (1 eq) in methanolTo the suspension was added 7M aqueous ammonia in methanol (7 eq). The reaction mixture was stirred at room temperature for 12 hours, then the reaction mixture was diluted with water and then filtered to give an off-white solid. The resulting solid was dissolved in ethyl acetate and washed with 1N hydrochloric acid solution. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 5-95% acetonitrile in water to afford the desired compound (4.1mg, 2% yield) as a white solid. ¹H-NMR(500MHz,CD₃OD) delta ppm 8.78(d,1H),8.69(d,1H),8.13(d,1H),7.54(s,1H), 7.26-7.31 (m,1H), 7.06-7.12 (m,1H), 7.03-7.06 (m,1H), 6.87-6.92 (m,2H,2 overlay shift), 5.98(s,2H),3.31(s,2H, and CD₃OD peak sync).

Compound 27

The title compound was prepared from intermediate-2 following general procedure C using 3-hydroxyisoxazole-5-carboxylic acid (1.3 eq) as the acidic reactant. 2.5 equivalents of T3P were used, and extraction with ethyl acetate was used during work-up. The crude product was purified by reverse phase HPLC eluting with a 5-95% acetonitrile/water gradient to give the desired compound (5.8mg, 5% yield) as a white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 11.90(br.s,1H),11.65(s,1H),9.12(d,1H),8.84(d,1H),8.09(d,1H),7.71(s,1H),7.32–7.36(m,1H),7.28(d,1H),7.22–7.26(m,1H),7.21(s,1H),7.10–7.13(m,1H),6.86–6.90(m,1H),5.94(s,2H)。

Compound 45

To a suspension of 2- (3-hydroxy-1H-pyrazol-4-yl) acetic acid (1 eq) in dichloromethane was added acetic anhydride (2 eq) at room temperature, followed by triethylamine (2 eq). The reaction mixture was stirred for 1 hour, then intermediate-2 (1 eq), triethylamine (2 eq), and a 50% ethyl acetate solution of 2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphospha-cyclohexane 2,4, 6-trioxide (1.15 eq) were added. The reaction was stirred at 50 ℃ for 6 hours and then at room temperature for another 12 hours, after which the reaction mixture was diluted with 1N hydrochloric acid solution and extracted with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel eluting with a gradient of 3-30% 7:1 acetonitrile/methanol in dichloromethane followed by a gradient to 15% methanol in dichloromethane to afford the desired intermediate, 1H-pyrazol-3-yl 4- (2- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-oxoethyl) acetate and the closely present impurity 1-acetyl-4- (2- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -oxy Ethyl) -1H-pyrazol-3-yl acetate (32.1mg, 33% yield) was an off-white solid.

To this acetate (1 eq) in tetrahydrofuran/water was added 1M aqueous sodium hydroxide (2 eq). The reaction mixture was stirred at 23 ℃ for 24 hours before adding additional sodium hydroxide (2 equivalents). After stirring for 36 hours, additional sodium hydroxide solution (2 equivalents) was added. After 40 hours, the reaction mixture was concentrated in vacuo to remove tetrahydrofuran and acidified by addition of 1M hydrochloric acid solution to form a precipitate. The solid was filtered and dried to give the desired compound (12.9mg, 44% yield) as an off-white solid.¹H-NMR(500MHz,CD₃OD) δ ppm 8.78(s,1H),8.67(d,1H),8.12(d,1H),7.54(s,1H),7.45(s,1H), 7.26-7.30 (m,1H), 7.08-7.12 (m,1H), 7.03-7.07 (m,1H), 6.86-6.90 (m,2H,2 overlay shift), 5.97(s,2H),3.58(s, 2H).

Compound 48

To a suspension of 2, 2-dimethylpropionamide (1 equivalent) in tetrahydrofuran was added a 1M solution of potassium bis (trimethylsilyl) amide (1 equivalent) in tetrahydrofuran. The reaction was stirred for 30 minutes, then concentrated in vacuo to give a brown solid. To the solid was added the intermediate-1A dimethylideneSulfone solution, and the reaction was stirred at 23 ℃. After 10 minutes, the reaction was diluted with 3M hydrochloric acid solution, filtered and dried to give a solid. The crude product was purified by reverse phase HPLC eluting with a 10-95% acetonitrile/water gradient to give a mixture of the two products. The mixture was further purified by silica gel chromatography, eluting with a gradient of 3-8% methanol in dichloromethane, to give the desired compound (2.2mg, 4% yield) as a white solid. ¹H-NMR(500MHz,CDCl₃) δ ppm 10.35(br.s,1H),8.55(d,1H),7.94(d,1H), 7.30-7.34 (m,1H),7.26(s,1H), 7.08-7.11 (m,3H),6.63(d,1H),5.91(s,2H),1.26(s, 6H); no 2N-H protons were observed.

Compound 66

Following general procedure C, the title compound was prepared from intermediate-2 using 3-ethoxy-2, 2-dimethyl-3-oxopropanoic acid (1 equivalent) as the acidic reactant, 1.5 equivalents of T3P, and dichloromethane for extraction. The crude product was chromatographed on silica gel with a gradient of 3-10% methanol in dichloromethane to give the desired compound (54.0mg, 42% yield) as a gummy solid.¹H-NMR(400MHz,CDCl₃)δppm 8.92(br.s,1H),8.74(d,1H),8.47(s,1H),8.13(d,1H),7.47(s,1H),7.27(s,1H),7.18–7.24(m,1H),7.02–7.06(m,1H),6.96–7.01(m,1H),6.80–6.85(m,1H),6.04(s,2H),4.26(q,2H),1.58(s,6H),1.30(t,3H)。

Compound 49

To compound 66(1 eq) in tetrahydrofuran/water was added 1M aqueous sodium hydroxide (1.08 eq). The reaction was stirred at 23 ℃ for 1.5 hours, then the reaction mixture was concentrated to remove tetrahydrofuran, and then acidified by adding 1M aqueous hydrochloric acid. The resulting precipitate was filtered and dried to obtain the desired compound (36.2mg, yield 75%) as light brownA colored solid.¹H-NMR(500MHz,DMSO-d₆) δ ppm 10.84(s,1H),9.11(d,1H),8.74(d,1H),8.01(d,1H),7.67(s,1H), 7.31-7.36 (m,1H),7.27(d,1H), 7.21-7.25 (m,1H), 7.10-7.13 (m,1H), 6.83-6.86 (m,1H),5.93(s,2H),1.44(s, 6H); no 1N-H protons were observed.

Compound 51

To a solution of compound 49(1 eq) in dichloromethane was added a solution of 2M oxalyl chloride (2.5 eq) in dichloromethane at 0 ℃. The reaction was stirred at 0 ℃ for 15 minutes and then warmed to 23 ℃. After 1 hour, the reaction mixture was concentrated in vacuo, tetrahydrofuran was added, and treated with saturated ammonium hydroxide solution. After 2 hours, the reaction was diluted with water, extracted with ethyl acetate, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 5-95% acetonitrile in water to afford the desired compound (9.2mg, 29% yield) as an off white solid.¹H-NMR(500MHz,CDCl₃)δppm 9.63(br.s,1H),8.79(d,1H),8.49(s,1H),8.13(s,1H),7.49(s,1H),7.20–7.25(m,1H),7.02–7.06(m,1H),6.98–7.02(m,1H),6.88–6.93(m,1H),6.64(d,1H),6.21(br.s,1H),6.03(s,2H),5.78(br.s,1H),1.26(s,6H).

Compound 35

To a suspension of 2-methylmalonic acid (1.0 eq) in dichloromethane was added oxalyl chloride (2.9 eq) at 0 ℃ followed by 3 drops of N, N-dimethylformamide. The reaction was stirred at 0 ℃ for 1 hour, after which the reaction was warmed to 23 ℃ and stirred for 30 minutes. The reaction mixture was concentrated in vacuo to give a brown oil, which was used in the subsequent step without purification.

To a suspension of intermediate-2 (1 equivalent) in dichloromethane was added 2-methylmalonyl dichloride (1.5 equivalents) followed by triethylamine (1.1 equivalents). Will be provided withThe reaction was stirred for 2 hours, after which the reaction was quenched by addition of saturated ammonium hydroxide. After stirring for a further 2 hours, the reaction mixture was diluted with 1M hydrochloric acid solution, extracted with ethyl acetate, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a 10-90% acetonitrile/water gradient to give the desired compound (3.3mg, 3% yield) as a white solid. ¹H-NMR(500MHz,CDCl₃)δppm 9.66(br.s,1H),8.77(d,1H),8.50(d,1H),8.11(d,1H),7.48(s,1H),7.21–7.26(m,1H),7.03–7.08(m,1H),6.99–7.03(m,1H),6.90–6.93(m,1H),6.64(d,1H),6.36(br.s,1H),6.13(br.s,1H),6.02(s,2H),3.44(q,1H),1.61(d,3H)。

Compound 36

The target compound is synthesized through 3 steps:

step 1: synthesis of ethyl methyl 3- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-methyl-3-oxopropanoate

To a solution of intermediate-2 (1 eq) and 3-methoxy-2-methyl-3-oxopropanoic acid (1.3 eq) in N, N-dimethylformamide was added triethylamine (4 eq) followed by a solution of 50% 1-propanephosphonic acid cyclic anhydride in ethyl acetate (2.5 eq). After 24 hours, the reaction mixture was diluted with water to form a tan precipitate. The solid was filtered and dried to give the desired intermediate, ethyl methyl 3- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-methyl-3-oxopropanoate (246mg, 93% yield) as a tan solid.¹H-NMR(500MHz,CDCl₃)δppm8.91(br.s,1H),8.75(d,1H),8.48(d,1H),8.10(d,1H),7.47(s,1H),7.19–7.23(m,1H),7.03–7.07(m,1H),6.96–7.00(m,1H),6.81–6.84(m,1H),6.62(d,1H),6.04(s,2H),3.81(s,3H),3.52(q,1H),1.56(d,3H)。

Step 2: synthesis of 1- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3-methoxy-2-methyl-benzoic acid 1, 3-dioxopropan-2-yl ester

To a solution of ethyl 3- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -pyrimidin-4-yl) amino) -2-methyl-3-oxopropanoate (1 eq) in tetrahydrofuran at 0 ℃ was added a solution of 1M sodium bis (trimethylsilyl) amide (1 eq) in tetrahydrofuran. After stirring for 20 minutes at 0 ℃, a solution of benzoyl peroxide (1.05 eq) in tetrahydrofuran was added. The reaction mixture was warmed to 23 ℃ and stirred for 12 hours. The reaction mixture was then diluted with saturated ammonium chloride solution, extracted with ethyl acetate, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a 5-95% acetonitrile/water gradient to give the desired intermediate, 1- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3-methoxy-2-methyl-benzoic acid 1, 3-dioxopropan-2-yl ester (48.3mg, 73% yield) as an off-white solid. ¹H-NMR(500MHz,CDCl₃)δppm 11.23(br.s,1H),10.59(s,1H),8.84(d,1H),8.51(d,1H),8.21(d,1H),8.13(d,1H),7.64–7.68(m,1H),7.50–7.54(m,3H),7.21–7.26(m,1H),7.01–7.06(m,3H),6.65(m,1H),6.00(s,2H),3.87(s,3H),2.01(s,3H)。

Step 3: synthesis of Compound 36

A solution of 1- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3-methoxy-2-methyl-1, 3-dioxopropan-2-benzoic acid ester (1 eq) in 1:1 tetrahydrofuran/saturated ammonium hydroxide solution was stirred at 23 ℃. After 3 hours, the reaction mixture was diluted with water, acidified by addition of 1N hydrochloric acid solution, extracted with ethyl acetate, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 5-95% acetonitrile in water to give the desired compound (13mg, 45% yield) asA white solid.¹H-NMR(500MHz,CD₃OD)δppm 8.79(s,1H),8.74(d,1H),8.17(d,1H),7.57(s,1H),7.27–7.31(m,1H),7.09–7.12(m,1H),7.04–7.07(m,1H),6.92(s,1H),6.89–6.92(m,1H),6.00(s,2H),1.70(s,3H)。

Compound 38 and compound 39

To a solution of intermediate-1A (1 eq) and 2- (3-oxo-2, 3-dihydro-1H-pyrazol-4-yl) acetic acid (1.2 eq) in 1, 4-dioxane was added triethylamine (4 eq). The reaction mixture was heated to 70 ℃ for 88 hours, then diluted with water and 1N hydrochloric acid solution and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a 5-95% acetonitrile/water gradient to afford the desired compound as a white solid, compound 38(20.5mg, 25% yield) and compound 39(6.1mg, 8% yield).

Compound 38:¹H-NMR(500MHz,DMSO-d₆)δppm 12.43(s,1H),11.24(s,1H),9.12(d,1H),8.89(d,1H),8.51(s,1H),7.74(s,1H),7.32–7.36(m,1H),7.27(d,1H),7.21–7.26(m,1H),7.10–7.14(m,1H),6.86–6.90(m,1H),5.95(s,2H),3.42(s,2H)。

compound 39:¹H-NMR(500MHz,CD₃OD) δ ppm 8.74(d,1H),8.68(d,1H),7.72(s,1H), 7.24-7.28 (m,1H),7.21(s,1H), 7.06-7.10 (m,1H), 7.01-7.06 (m,1H), 6.83-6.86 (m,2H,2 overlay shift), 5.92(s,2H),3.44(s, 2H).

Compound 40

To a suspension of compound 39(1 eq) in dichloromethane was added HATU (1.2 eq), N-ethyl-N-isopropylpropan-2-amine (3 eq) and 0.5M ammonia in dioxane (6 eq). After 2 hours, the reaction mixture was concentrated in vacuo. The crude product was passed through reverse phase HPLPurification by elution with a 5-95% acetonitrile/water gradient afforded the desired compound, compound 40(0.7mg, 8% yield) as a white solid.¹H-NMR(500MHz,CD₃OD)δppm 8.74(s,1H),8.69(br.s,1H),7.72(s,1H),7.26–7.29(m,1H),7.25(s,1H),7.05–7.10(m,1H),7.01–7.05(m,1H),6.87(s,1H),6.83–6.86(m,1H),5.92(s,2H),3.34(s,2H)。

Compound 56 and compound 57

To a suspension of compound 38(1 equivalent) in diethyl ether/methanol (1:1) was added a 2M solution of trimethylsilyldiazomethane (1.5 equivalents) in diethyl ether. After completion of the reaction, the reaction mixture was concentrated in vacuo to give a mixture of esters (19.2mg, 0.038mmol) as a yellow residue which was used in the next step without purification.

To the mixture of esters (1 eq) was added 7M ammonia in methanol (222 eq). The reaction mixture was stirred at room temperature for 12 hours, then concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a 5-95% acetonitrile/water gradient to afford the two desired compounds, compound 56(2.9mg, 16% yield) and compound 57(0.9mg, 5% yield) as white solids.

Compound 56:¹H-NMR(500MHz,CD₃OD) δ ppm 8.79(m,2H,2 shift overlap), 8.28(s,1H),7.53(s,1H), 7.26-7.30 (m,1H), 7.08-7.11 (m,1H), 7.03-7.06 (m,1H),6.96(s,1H), 6.90-6.94 (m,1H),5.98(s,2H),3.72(s,3H),3.34(s, 2H).

Compound 57:¹H-NMR(500MHz,CDCl₃)δppm 8.71(d,1H),8.56(s,1H),8.51(s,1H),7.44(s,1H),7.24–7.27(m,1H),7.05–7.08(m,1H),7.00–7.05(m,1H),6.88–6.93(m,1H),6.67(s,1H),6.04(s,2H),5.83(br.s,1H),5.43(br.s,1H),4.12(s,3H),3.47(s,2H)。

compound 41

To a solution of compound 38 and compound 39(1 equivalent) in dichloromethane/acetonitrile (1:1) was added HATU (1.2 equivalents), N-ethyl-N-isopropylpropan-2-amine (3 equivalents), 4-dimethylaminopyridine (0.1 equivalent) and 0.5M ammonia in dioxane (6 equivalents). After 12 h, the reaction mixture was diluted with water and 1M hydrochloric acid solution, extracted with ethyl acetate, dried over sodium sulfate, filtered and concentrated in vacuo. Methanol was added to the mixture to give an off-white precipitate. The solid was further purified by reverse phase HPLC eluting with a gradient of 5-95% acetonitrile in water to afford the desired compound (4.1mg, 3% yield) as a white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 11.28(s,1H),9.12(s,1H),8.88(d,1H),8.47(s,1H),7.73(s,1H),7.48(br.s,1H),7.30–7.37(m,1H),7.27(s,1H),7.20–7.25(m,1H),7.09–7.14(m,1H),7.02(br.s,1H),6.85–6.91(m,1H),5.95(s,2H),3.26(s,2H)。

Compound 59

The title compound was prepared according to general procedure C using intermediate-2, using 3-methyl-2-oxopyrrolidine-3-carboxylic acid (1.05 eq) as the acidic reactant, 1.5 eq of T3P, stirring the reaction at 50 ℃ for 12 h, then at 80 ℃ for 36 h, and extraction with ethyl acetate during work-up. The crude product was purified by reverse phase HPLC eluting with a gradient of 10-95% acetonitrile in water to afford the desired compound (1.6mg, 2% yield) as an off white solid. ¹H-NMR(500MHz,CD₃OD)δppm 8.78(d,1H),8.71(d,1H),8.12(d,1H),7.56(s,1H),7.26–7.30(m,1H),7.08–7.12(m,1H),7.03–7.07(m,1H),6.93(d,1H),6.86–6.90(m,1H),5.99(s,2H),3.36–3.46(m,2H),2.72–2.78(m,1H),2.10–2.15(m,1H),1.58(s,3H)。

Compound 3

A suspension of intermediate-7 (1 eq) in phosphoryl chloride (62 eq) was heated at 90 ℃ for 2 hours, after which the reaction mixture was concentrated in vacuo to give the desired chloro intermediate, 4-chloro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5, 7-dihydrothieno [3,4-d]The dioxide (155mg, 100% yield) was a tan solid. To a solution of the chloro intermediate (1 eq) in tetrahydrofuran was added acetic acid (2 eq) and zinc powder (2 eq). The resulting suspension was heated at 70 ℃ for 24 hours, after which the reaction was filtered through celite, diluted with saturated ammonium chloride solution and extracted with ethyl acetate. The combined organic layers were washed with water, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a 5-95% acetonitrile/water gradient to give the desired compound, compound 3(4.0mg, 4% yield) as an off-white solid.¹H-NMR(500MHz,CDCl₃)δppm 8.80(s,1H),8.49(d,1H),7.49(s,1H),7.20–7.25(m,1H),7.03–7.07(m,1H),6.97–7.00(m,1H),6.83–6.86(m,1H),6.61(d,1H),6.05(s,2H),4.56(s,2H),4.51(s,2H)。

Compound 75

To a solution of 2- (5-hydroxyisoxazol-3-yl) acetic acid (2.0 eq) in acetonitrile was added acetic anhydride (2.0 eq) followed by triethylamine (2.0 eq). After 30 minutes, intermediate-2 (1.0 equiv.) was added followed by additional triethylamine (2.0 equiv.) and a 50% solution of 1-propanephosphonic acid cyclic anhydride in ethyl acetate (2.0 equiv.). The reaction was stirred at room temperature for 24 hours, then the reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 3-10% methanol in dichloromethane to give the desired intermediate, 3- (2- ((2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-oxoethyl) isoxazol-5-yl acetate (34.1mg, 9% yield) as a cream solid.

To 3- (2- ((2)- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-oxoethyl) isoxazol-5-yl acetate (1.0 eq) in tetrahydrofuran/water was added 1M aqueous sodium hydroxide solution (3.0 eq). After 5 min, the reaction mixture was quenched by addition of 1M hydrochloric acid solution and concentrated in vacuo. The crude product was purified by reverse phase HPLC eluting with a 10-95% acetonitrile/water gradient to give the desired compound (2.1mg, 7% yield) as a white solid.¹H-NMR (500MHz, acetone-d)₆) δ ppm 10.29(s,1H),8.92(s,1H),8.75(d,1H),8.03(d,1H),7.55(d,1H), 7.32-7.37 (m,1H), 7.16-7.20 (m,1H), 7.09-7.13 (m,2H,2 overlay shift), 6.94-6.98 (m,1H),6.01(s,2H),4.00(s,2H),3.88(s,1H),2.53(s, 1H).

Compound 11

To a solution of 5- (trifluoromethyl) -1,3, 4-thiadiazol-2-amine (1 eq) and intermediate-1A (1 eq) in DMF was added Cs₂CO₃(3 eq.) the mixture was stirred at 90 ℃ for 24 h. The reaction was cooled to 23 ℃ and diluted with ethyl acetate. The organic phase was washed with water and brine, concentrated in vacuo, and purified by reverse phase HPLC to give the desired compound, compound 11(8mg, 13% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.15-9.17(m,1H),8.76(d,1H),7.64(s,1H),7.32-7.40(m,2H),7.30(d,1H),7.17-7.25(m,2H),7.09-7.15(m,1H),5.95(s,2H)。

Compound 12

To a solution of intermediate-1A (1 eq) in DMF/water (2:3) was added Cs ₂CO₃(2 equivalents) and the resulting mixture was stirred at 80 ℃ for 2 hours. The reaction was cooled to 23 ℃, the solid was filtered and dried under high vacuum. The residue was then purified by silica gel chromatography (ethyl acetate in hexane, 5-35% gradient) to afford the desired compoundMaterial (15mg, 21% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.11-9.16(m,2H),8.29(s,2H),7.73(s,1H),7.33-7.39(m,1H),7.32(d,1H),7.22-7.28(m,1H),7.15(t,1H),7.03(t,1H),5.94(s,2H)。

Compound 13

To a solution of 5- (trifluoromethyl) -1,3, 4-oxadiazol-2-amine (2 eq) in THF was added LiHMDS (2 eq) and the resulting mixture was stirred at 0 ℃ for 10 min. A solution of intermediate-1A (1 eq) in THF was then added and the mixture was stirred overnight. The solvent was removed in vacuo and the resulting residue was purified by reverse phase HPLC to give the desired compound (5mg, 7% yield) as a white solid.¹H NMR(500MHz,CDCl₃)δppm 8.85(br.s.,1H),8.56(d,1H),8.53(d,1H),7.25-7.34(m,4H),7.03-7.13(m,3H),6.63(d,1H),5.93(s,2H)。

Compound 14

To a solution of 5-methyl-1, 3, 4-oxadiazol-2-amine (2 equiv.) and intermediate-1A (1 equiv.) in DMF was added Cs₂CO₃(3 equivalents). The mixture was stirred at 80 ℃ for 4 hours and then filtered. The filtrate was concentrated in vacuo, and the residue was purified by reverse phase HPLC to give the desired compound (22mg, 36% yield) as a brown solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.12(d,1H),8.62(br.s.,1H),7.56(br.s.,1H),7.31-7.38(m,1H),7.19-7.27(m,2H),7.12(t,1H),6.96(t,1H),5.92(s,2H),2.48(s,3H)。

Compound 15

To a solution of intermediate-8 (1 eq) in dichloromethane was added HATU (1.1 eq) and N-ethylThe radical-N-isopropylpropan-2-amine (3 equivalents). The mixture was stirred at 0 ℃ for 15 minutes, then warmed to 23 ℃ and stirred for an additional 1 hour. Ammonia (5 equivalents) was added to the reaction and the mixture was stirred at 23 ℃ for 4 hours. The reaction was diluted with saturated NH 4Cl solution and water and extracted 3 times with ethyl acetate. The organic layer was washed 2 times with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by reverse phase HPLC to give the desired compound (16mg, yield 30%) as a white solid. ¹H NMR(500MHz,CD₃OD)δppm 8.80(d,1H),8.31(d,1H),7.61-7.67(m,1H),7.24-7.34(m,1H),7.02-7.14(m,2H),6.97(d,1H),6.92(t,1H),6.01(s,2H),4.42(t,2H)。

Compound 16

The title compound is prepared according to general method B starting from intermediate-1A, using isoxazolidine hydrochloride as amine reactant and heating a dioxane/water (10:1) solution of the reactant at 110 ℃ for 24 h. The reaction was cooled to 23 ℃, the solvent was removed in vacuo, and the resulting residue was purified by reverse phase HPLC to give the desired compound (38mg, 69% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 9.03(d,1H),8.44(d,1H),7.54(s,1H),7.23-7.29(m,1H),7.20(d,1H),7.13-7.19(m,1H),7.04(t,1H),6.77(t,1H),5.86(s,2H),3.95(t,2H),3.83-3.89(m,2H),2.23(q,2H)。

Compound 34

The title compound was prepared according to general procedure B using 1, 2-oxazinane hydrochloride as the amine reactant and a dioxane/water (10:1) solution of the reactant was heated at 110 ℃ for 24 hours. The reaction was cooled to 23 ℃, the solvent was removed in vacuo, and the resulting residue was purified by reverse phase HPLC to give the desired compound (36mg, 63% yield) as a white solid.¹H NMR(400MHz,DMSO-d₆)δppm 9.03(d,1H),8.41(d,1H),7.56(s,1H),7.22-7.30(m,1H),7.20(d,1H),7.15(dd,1H),7.00-7.07(m,1H),6.77(t,1H),5.86(s,2H),4.02(br.s.,2H),3.90(br.s.,2H),1.73(br.s.,4H)。

Compound 42

Step 1: synthesis of ethyl methyl 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionate

To the intermediate-4 (1 eq) in THF suspension was added (diazomethyl) trimethylsilane (2 eq) and the mixture was stirred at 80 ℃ for 4 hours. The reaction was cooled to 23 ℃ and the solvent was removed in vacuo to give the desired intermediate, ethyl methyl 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionate (80mg, 97% yield) as a brown solid.

Step 2: synthesis of 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide

To a solution of ethyl methyl 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionate (1 eq) in ethanol was added hydrazine hydrate (15 eq) and the mixture was stirred overnight. The solvent was removed in vacuo and the residue was slurried with hexane and filtered. The resulting solid was dried under high vacuum to give the desired intermediate, 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (80mg, yield 100%) as a white solid.

Step 3: 2- (3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (iso-butyl)) phenylSynthesis of oxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionyl) hydrazinothioamide

Trimethylsilazane isothiocyanate (2 eq) was added to a solution of 3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropanohydrazide (1 eq) in isopropanol. The mixture was heated at 90 ℃ for 3 hours, cooled to 23 ℃ and filtered. The solid was collected and dried under high vacuum to give the desired intermediate, 2- (3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionyl) hydrazinothioamide (80mg, 90% yield) as a white solid.

Step 4: synthesis of Compound 42

To a solution of 2- (3,3, 3-trifluoro-2- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxypropionyl) hydrazinothioamide (1 eq) in THF was added 4-methylbenzene-1-sulfonyl chloride (1.5 eq) and pyridine (2 eq). The mixture was heated by microwave at 150 ℃ for 30 minutes. The solvent was removed and the residue was purified by reverse phase HPLC to give the desired compound (5.5mg, yield 28%) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.71(s,1H),8.20(d,1H),7.35(s,1H),7.16-7.24(m,1H),6.93-7.05(m,2H),6.81-6.89(m,2H),5.90(s,2H),4.40(d,1H),4.21(d,1H)。

Compound 43

To intermediate-1A (1 eq.) and 5,6,7, 8-tetrahydro- [1,2,4 ]]Triazolo compounds[4,3-a]1 equivalent of pyrazin-3-ol) in DMF was added Cs₂CO₃(2 eq.) and the mixture was stirred at 80 ℃ for 2 hours. The reaction was cooled to 23 ℃, filtered, and the filtrate was concentrated in vacuo. The residue was purified by reverse phase HPLC to give the desired compound (5mg, 7% yield) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.95(d,1H),8.77-8.80(m,1H),7.58(s,1H),7.24-7.31(m,1H),7.07-7.13(m,1H),7.01-7.07(m,1H),6.85-6.91(m,2H),5.98(s,2H),4.48(s,2H),3.97(t,2H),3.69-3.75(m,2H)。

Compound 44

To intermediate-1A (1 eq.) and 5,6,7, 8-tetrahydro- [1,2,4 ]]Triazolo [4,3-a]Adding Cs into a solution of pyrazine-3-alcohol (1 equivalent) in DMF₂CO₃(2 eq.) and the mixture was stirred at 80 ℃ for 2 hours. The reaction was cooled to 23 ℃, filtered, and the filtrate was concentrated in vacuo. The residue was purified by reverse phase HPLC to give the desired compound (6mg, yield 8%) as a white solid. ¹H NMR(500MHz,DMSO-d₆)δppm 11.64(s,1H),9.08-9.12(m,1H),8.43(d,1H),7.63(s,1H),7.30-7.36(m,1H),7.26(d,1H),7.19-7.25(m,1H),7.11(t,1H),6.84(t,1H),5.85-5.97(m,2H),4.90(s,2H),4.17(t,2H),3.68(t,2H)。

Compound 65

To a solution of intermediate-4 (1 eq) in dichloromethane was added PyAOP (2 eq) and N-ethyl-N-isopropylpropan-2-amine (2 eq) and the mixture was stirred for 30 min. Cyclopropylamine (1.5 eq) was added to the reaction and the reaction was stirred for an additional 24 hours. The solvent was removed in vacuo and the residue was purified by HPLC to give the desired compound (5mg, 22% yield) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.80(d,1H),8.25(d,1H),7.54(s,1H),7.26-7.33(m,1H),7.02-7.13(m,2H),6.92-6.97(m,2H),5.99(s,2H),4.35(d,1H),4.07(d,1H),2.61(br,1H),0.51-0.72(m,2H),0.29-0.49(m,2H)。

Compound 76

The title compound was prepared according to general procedure B using intermediate-1B (1 equivalent) instead of intermediate-1A, 2-aminoethanesulfonamide (1.5 equivalents) as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 12 h. The crude product was purified by reverse phase HPLC to give the desired compound (12mg, 48% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm8.25(d,1H),7.99(br.s.,1H),7.73(s,1H),7.30-7.37(m,1H),7.20-7.26(m,1H),7.11(t,1H),7.03(s,2H),6.84(t,1H),5.83(s,2H),3.84-3.91(m,2H),3.34(t,2H),2.56(s,3H)。

Compound 77

The title compound was prepared according to general procedure B using intermediate-1B (1 equivalent) instead of intermediate-1A, piperazin-2-one (1.5 equivalents) as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 12 hours. The resulting crude product was purified by reverse phase HPLC to give the desired compound (13mg, 55% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm8.36(d,1H),8.22(br.s.,1H),7.75(s,1H),7.33(q,1H),7.19-7.26(m,1H),7.11(t,1H),6.78-6.84(m,1H),5.83(s,2H),4.33(s,2H),3.98(t,2H),3.35(br.s,2H),2.58(s,3H)。

Compound 78

The title compound was prepared according to general procedure B using intermediate-1B (1 equivalent) instead of intermediate-1A and thiomorpholine 1, 1-dioxide (1.5 equivalents) Amount) as amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 12 hours. The resulting crude product was purified by reverse phase HPLC to give the desired compound (11mg, 43% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.42(d,1H),7.76(s,1H),7.33(q,1H),7.19-7.26(m,1H),7.11(t,1H),6.81(t,1H),5.83(s,2H),4.21(br.s.,4H),3.33(br.s.,4H),2.58(s,3H)。

Compound 79

The title compound was prepared according to general procedure B using intermediate-1B (1 equivalent) instead of intermediate-1A, 3-difluoropiperidine (1.5 equivalents) as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 12 hours. The resulting crude product was purified by reverse phase HPLC to give the desired compound (14mg, 56% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.38(d,1H),7.75(s,1H),7.33(q,1H),7.20-7.26(m,1H),7.11(t,1H),6.82(t,1H),5.83(s,2H),4.14(t,2H),3.82-3.88(m,2H),2.58(s,3H),2.10-2.21(m,2H),1.81(br.s.,2H)。

Compound 80

The title compound was prepared according to general procedure B using intermediate-1B (1 equivalent) instead of intermediate-1A, 3-methoxypyrrolidine (1.5 equivalents) as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 12 hours. The crude product was purified by reverse phase HPLC to give the desired compound (12mg, 51% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.27(d,1H),7.73(s,1H),7.30-7.37(m,1H),7.20-7.26(m,1H),7.09-7.14(m,1H),6.82(t,1H),5.83(s,2H),4.09(br.s.,1H),3.64-3.91(m,4H),3.28(s,3H),2.58(s,3H),1.97-2.15(m,2H)。

Compound 1

The target compound is prepared through 5 steps:

step 1: synthesis of ethyl 5- ((bis- (tert-butoxycarbonyl)) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate

To a solution of 4-dimethylaminopyridine (0.05 eq), 5-amino-1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylic acid ethyl ester (1 eq) and triethylamine (2 eq) in tetrahydrofuran was added Boc anhydride (1.5 eq). After stirring for 96 hours, the solution was poured into ethyl acetate and water. The layers were separated and the organic layer was washed with 5% potassium hydrogen sulfate solution (3 times), saturated sodium bicarbonate solution and saturated aqueous sodium chloride solution. The solution was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude product was combined with the crude product from the previous reaction and purified by silica gel chromatography (0-50% ethyl acetate in hexanes) to give the desired intermediate, ethyl 5- ((bis- (tert-butoxycarbonyl)) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (236mg, 63% total yield) as an oil.

Step 2: synthesis of ethyl 5- ((tert-butoxycarbonyl) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate

A suspension of potassium carbonate (3 equivalents) and methyl 5- ((bis- (tert-butoxycarbonyl)) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (1 equivalent) in ethanol was heated at 60 ℃ for 3 hours. The solvent was removed in vacuo and the residue diluted with dichloromethane and saturated aqueous ammonium chloride (1: 1). The layers were separated and the aqueous layer was extracted 2 times with dichloromethane. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (0-35% ethyl acetate in hexanes) afforded the desired intermediate, ethyl 5- ((tert-butoxycarbonyl) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (137mg, 79% yield) as a clear oil.

Step 3: 5- ((tert-Butoxycarbonyl)Synthesis of ethyl (methyl) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate

To a solution of ethyl 5- ((tert-butoxycarbonyl) amino) -1- (2-fluorobenzyl) -1-H-pyrazole-3-carboxylate (1 eq) in N, N-dimethylformamide at 0 ℃ was added sodium hydride (1.5 eq) as a 60% dispersion in mineral oil. After stirring for 15 minutes at 0 ℃, methyl iodide (2.5 equivalents) was added in one portion. The solution was immediately warmed to 23 ℃. After 30 minutes, the solution was cooled to 0 ℃ and saturated aqueous ammonium chloride solution was added. The solution was then warmed to 23 ℃ and diluted with ethyl acetate and water (1: 1). The layers were separated and the aqueous layer was extracted 2 times with ethyl acetate. The organics were washed with water (3 times) and saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, and the solvent removed in vacuo. The crude product was combined with the crude product from the previous reaction and purified by silica gel chromatography (ethyl acetate in hexanes) to give the desired intermediate, ethyl 5- ((tert-butoxycarbonyl) (methyl) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (105mg, 76% total yield) as a clear oil.

Step 4：

Synthesis of 1- (2-fluorobenzyl) -5- (methylamino) -1H-pyrazole-3-carboxamidine

To a toluene suspension of ammonium chloride (5.5 equivalents) was added dropwise a 2M solution of trimethylaluminum (5 equivalents) in heptane over 5 minutes. After stirring for 30 minutes, the bubbling was reduced and the aluminum reagent was added to a solution of ethyl 5- ((tert-butoxycarbonyl) (methyl) amino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (1 eq) in toluene. The solution was heated to 90 ℃ and reacted at this temperature for 20 hours. The solution was then cooled to 0 ℃ and methanol (10 equivalents) was added. The solution was immediately removed from the ice bath and warmed to 23 ℃. After stirring for 30 min, the suspension was filtered through celite, washing with methanol to give the desired intermediate, 1- (2-fluorobenzyl) -5- (methylamino) -1H-pyrazole-3-carboxamidine (43mg, 55% yield) as a white solid.

Step 5: synthesis of Compound 1

To a suspension of 1- (2-fluorobenzyl) -5- (methylamino) -1H-pyrazole-3-carboxamidine (1 eq) in ethanol was added sodium 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (3 eq). The solution was stirred in a sealed vial at 90 ℃ for 16 hours. Then adding 1, 8-diazabicyclo [5.4.0 ]]Undec-7-ene (200. mu.L), and the resulting solution was stirred at 90 ℃ for 17 hours. The solvent was removed in vacuo and purified by silica gel chromatography (gradient elution with 0-10% methanol in dichloromethane) to give the desired compound, compound 1(1.2mg, 2% yield) as a yellow film. ¹H-NMR(500MHz,CD₃OD)δppm 7.99(br s,1H),7.35-7.31(m,1H),7.16-7.11(m,2H),6.94-6.91(m,1H),6.10(s,1H),5.35(s,2H),2.84(s,3H)。

Compound 81

The target compound is synthesized through 3 steps:

step 1: synthesis of ethyl 5- (dimethylamino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate

To a solution of ethyl 5-amino-1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (1 eq) in N, N-dimethylformamide at 0 ℃ was added sodium hydride (2.5 eq) as a 60% dispersion in mineral oil. The resulting suspension was stirred at 0 ℃ for 20 minutes, then methyl iodide (3 equivalents) was added and the solution was immediately warmed to 23 ℃. After stirring for 1.25 hours, the solution was cooled to 0 ℃ and saturated ammonium chloride was added. After warming to 23 ℃, the suspension was diluted with ethyl acetate and water (1: 1). The layers were separated and the aqueous layer was extracted with ethyl acetate. The organics were washed with water (3 times) and brine, dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (0-20% ethyl acetate in hexane) to give the desired intermediate, ethyl 5- (dimethylamino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (69mg, 62% yield) as a clear oil.

Step 2: synthesis of 5- (dimethylamino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxamidine

A 2M solution of trimethylaluminum (5 equivalents) in heptane was added dropwise to a toluene suspension of ammonium chloride (5.5 equivalents) over 2 minutes. After stirring for 30 minutes, a solution of ethyl 5- (dimethylamino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxylate (1 eq) in toluene was added. The resulting solution was heated at 80 ℃ for 15 hours and then cooled to 0 ℃. Methanol (10 equivalents) was added and the reaction mixture was warmed to room temperature and stirred for 30 minutes. Additional methanol was added and the suspension was filtered through celite. The solvent was removed in vacuo to give the desired intermediate, 5- (dimethylamino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxamidine (51mg, 82% yield) as a yellow solid.

Step 3: synthesis of Compound 81

Sodium 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-olate (3 equiv.), 5- (dimethylamino) -1- (2-fluorobenzyl) -1H-pyrazole-3-carboxamidine (1 equiv.) and 1, 8-diazabicyclo [5.4.0]A solution of undec-7-ene (1 eq) in ethanol was heated at 90 ℃ for 2.5 h. The solvent was then removed in vacuo and the resulting residue was dissolved in dichloromethane and filtered. The resulting solid was suspended in dichloromethane and filtered again. The solutes from the two filtrations were combined and purified by column chromatography (methanol in dichloromethane) to give the desired compound (25mg, yield 39%) as a yellow solid.¹H-NMR(500MHz,CD₃OD)δppm 8.00(d,1H),7.36-7.32(m,1H),7.16-7.09(m,3H),6.62(s,1H),5.45(s,2H),2.70(s,6H)。

Compound 50

Following general procedure C, the title compound was prepared starting from intermediate-2, using 1- (methylsulfonyl) cyclopropanecarboxylic acid (2 equivalents) as the acidic reactant, 6 equivalents of triethylamine and 4 equivalents of propylphosphonic anhydride (T3P), and the solution was heated to 65 ℃ for 2 hours. The solution was poured into 1N aqueous hydrochloric acid and dichloromethane (1: 1). The layers were separated and the aqueous layer was extracted 2 times with dichloromethane. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (20-100% ethyl acetate in hexanes) gave the desired compound (30mg, 28% yield) as a white solid. ¹H-NMR(500MHz,CDCl₃)δppm 8.81(d,1H),8.50(d,1H),8.05(d,1H),7.54(s,1H),7.25-7.21(m,1H),7.07-7.04(m,1H),7.01-6.98(s,1H),6.90-6.87(m,1H),6.66(d,1H),6.05(s,2H),3.17(s,3H),1.89-1.83(m,4H)。

Compound 54

A solution of intermediate-9 (1 eq) in methanol was treated with a solution of 7N ammonia (100 eq) in methanol and heated at 50 ℃ for 1.5 hours. After storage at 0 ℃ overnight, an additional 7N ammonia (150 equivalents) in methanol was added and the solution was heated to 50 ℃ for 1 hour. An additional 125 equivalents of ammonia in methanol was added and the resulting solution was heated at 50 ℃ for 2 hours. After storage in a freezer for two night, the suspension was filtered and washed with dichloromethane to give the desired compound (8mg, 47% yield) as a white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.11(s,1H),8.44(d,1H),8.26(s,1H),7.82(s,1H),7.66(s,1H),7.35-7.31(m,1H),7.28(m,1H),7.24-7.21(m,1H),7.11(t,1H),6.85(t,1H),5.92(s,2H),5.23(s,2H),4.48-4.46(m,2H),4.23-4.21(m,2H)。

Compound 19

A solution of intermediate-16 (1 eq) and 3,3, 3-trifluoropropane-1-sulfonyl chloride (1.1 eq) in pyridine and dichloromethane (1:2) was stirred at 23 ℃ for 5 hours. 1N NaOH was then added to the reaction mixture and stirring was continued for 1.5 hours, after which it was diluted with dichloromethane and water and then acidified to pH 3 with 1N HCl solution. The phases were separated and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated. The crude product was purified by reverse phase HPLC (C18 column, eluting with a gradient of 30% to 60% acetonitrile in water containing 0.1% TFA over 20 min) to give the desired compound (11mg, 24% yield) as a yellow solid. ¹H NMR(500MHz,DMSO-d₆)δppm9.13(d,1H),8.83-8.93(m,1H),7.47(s,1H),7.32-7.38(m,1H),7.21-7.27(m,2H),7.11(t,1H),6.84(t,1H),5.94(s,2H),3.52-3.56(m,2H),2.71-2.79(m,2H)。

Compound 29

Phosphorus oxychloride (8 equivalents) was added dropwise via syringe to a solution of intermediate-11 (1 equivalent) in pyridine at 0 ℃. The reaction mixture was slowly warmed to 23 ℃ and stirred continuously for 2 hours. The volatiles were removed in vacuo and the residue was dissolved in ethyl acetate, washed with water, brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude solid was then purified by reverse phase HPLC (C18 column, eluting with a gradient of 20% to 60% acetonitrile in water containing 0.1% TFA, 20 min) to give the desired compound (10mg, 9% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 11.78(s,1H)9.10(d,1H)7.40(s,1H)7.30-7.37(m,1H)7.25-7.30(m,2H)7.23(d,2H)7.08-7.13(m,1H)6.84-6.89(m,1H)5.89(s,2H)1.80(s,3H)。

Compound 64

Following general procedure B, the title compound was prepared starting from intermediate-1A, using 2- (3-amino-1, 1, 1-trifluoro-2-hydroxypropan-2-yl) -1-methyl-1H-imidazole-1-chloride (1.5 equivalents) as the amine reactant and 5 equivalents of triethylamine, and the reactant was heated to 90 ℃ in dioxane/water (3:1) for 15 hours. The reaction was cooled to 23 ℃ and the volatiles were removed under nitrogen. The resulting crude product was purified by reverse phase HPLC (C18 column, eluting with a gradient of 5% to 95% acetonitrile in water containing 0.1% TFA over 20 minutes) to give the desired compound (32mg, 83% yield) as a white powder. ¹H NMR(500MHz,DMSO-d₆)δppm 9.11(s,1H),8.31(d,1H),7.95(br.s.,1H),7.52(s,1H),7.29-7.38(m,2H),7.19-7.26(m,2H),7.11(t,2H),6.94(t,1H),5.92(s,2H),4.25-4.40(m,2H),3.88(s,3H),2.54(s,1H).

Compound 32

Chloroacetyl chloride (3 equivalents) was added to a solution of intermediate-2 (1 equivalent) and triethylamine (5 equivalents) in dichloromethane at 0 ℃ and then warmed to 23 ℃ for 20 minutes. Adding saturated NaHCO₃And ethyl acetate/THF 1:1 quench the reaction. Filtration afforded the desired compound (28mg, 89%) as a brown solid.¹H NMR(500MHz,DMSO-d6)δppm 11.21-11.36(m,1H),9.11(d,1H),8.74(d,1H),8.56(t,1H),7.97(d,1H),7.66(s,1H),7.30-7.38(m,1H),7.27(d,1H),7.19-7.25(m,1H),7.12(t,1H),6.89(t,1H),5.93(s,2H),4.18(s,2H),4.01-4.10(m,2H)。

Intermediate-15

The target compound is synthesized through 3 steps:

step 1: (3- (trifluoromethyl) - [1,2,4]]Triazolo [4,3-a]Synthesis of pyrazin-6-yl) methanol

Trifluoroacetic anhydride (10 equivalents) was added to (5-hydrazinopyrazin-2-yl) methanol (1 equivalent) at 0 ℃. After the addition was complete, the reaction was warmed to 23 ℃, stirred for 20 minutes, and the solvent was removed in vacuo. Polyphosphoric acid (excess) was then added to the reaction and the reaction was heated at 100 ℃ for 2 hours. The hot suspension was poured onto ice and basified with ammonium hydroxide to pH 11. The mixture was extracted with ethyl acetate, concentrated and purified by silica gel chromatography to give the desired intermediate, (3- (trifluoromethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-6-yl) methanol (2.035g, 43%) as a yellow solid.

¹H NMR(500MHz,DMSO-d6)δppm 9.53-9.73(m,1H),8.32(d,1H),5.82(t,1H),4.70(dd,2H)。

Step 2: 6- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (trifluoromethyl) - [1,2,4]Triazolo [4,3-a ]Synthesis of pyrazines

Tert-butyldiphenylsilyl chloride (2 equivalents) was added to a solution of (3- (trifluoromethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-6-yl) methanol (1 equivalent) and imidazole (3 equivalents) in methylene chloride. The reaction was stirred at 23 ℃ for 20 minutes, quenched with water, extracted with ethyl acetate, concentrated, and purified by silica gel chromatography to give the desired intermediate, 6- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine (0.414g, 94%) as a yellow oil.

¹H NMR(500MHz,DMSO-d6)δppm 9.58-9.71(m,1H),8.44(s,1H),7.61-7.76(m,4H),7.34-7.53(m,6H),4.93(d,2H),1.00-1.12(m,9H)。

Step 3: 6- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4]Triazolo [4,3-a]Synthesis of pyrazines

Reacting 6- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (trifluoromethyl) - [1,2, 4%]Triazolo [4,3-a]Pyrazine (1 eq) was taken up in ethanol with 10% Pd/C (0.05 eq) and hydrogenated under hydrogen balloon pressure at 23 ℃ for 40 h. The reaction mixture was filtered, concentrated, and purified by silica gel chromatography to give the desired intermediate, 6- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4]Triazolo [4,3-a]Pyrazine (0.16g, 38%) as a brown solid. ¹H NMR(500MHz,DMSO-d6)δppm 7.63(ddd,4H),7.39-7.52(m,6H),4.18-4.28(m,2H),3.95-4.02(m,1H),3.84-3.91(m,1H),3.71-3.83(m,2H),3.23-3.32(m,1H),2.79(td,1H),1.00(s,9H)。

Step 4: synthesis of intermediate-15

Tetrabutylammonium fluoride (2 equiv.) was added to intermediate-1A (1 equiv.), 6- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4]Triazolo [4,3-a]Pyrazine (2 equiv.) and triethylamine (2 equiv.) were suspended in dioxane/water (10:1) and stirred at 100 ℃ for 48 h. The reaction mixture was then cooled to 23 ℃, concentrated in vacuo, and purified by reverse phase HPLC to give the desired compound intermediate-15 (6mg, 36%) as a white solid.¹H NMR(500MHz,DMSO-d6)δppm 9.11(d,1H),8.46(d,1H),7.67(s,1H),7.30-7.38(m,1H),7.28(d,1H),7.19-7.25(m,1H),7.11(t,1H),6.85(t,1H),5.88-5.98(m,2H),5.61(d,1H),5.20(t,1H),5.13(br.s.,1H),4.82(d,1H),4.38-4.51(m,2H),3.64(dt,1H),3.50-3.59(m,1H)。

Compound 62

Preparation of intermediate-15 (1 eq) and NMO (10 eq) at 23 deg.CTo a suspension of acetonitrile and water (10 equivalents) was added tetrapropylammonium ruthenate (0.1 equivalent). The reaction was monitored by LCMS for completion, filtered and purified by reverse phase HPLC to give the desired compound (9mg, 12%) as a white solid.¹H NMR(500MHz,CD₃OD) δ ppm 8.72-8.85(m,1H),8.50(d,1H),7.54-7.70(m,1H),7.20-7.36(m,1H),7.07-7.15(m,1H),7.04(t,1H),6.94(d,1H),6.86(t,1H),6.28(br.s.,1H),6.00(s,2H),5.70(d,1H),5.08-5.21(m,1H),5.01(d,1H),4.60(dd,1H). COOH proton exchange.

Compound 83

A solution of 2- (1- (2, 3-difluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -5-fluoropyrimidin-4-ol (described in the previous patent: WO2013/101830A1) (1 eq) and sodium methoxide in methanol (2.2 eq) was suspended in diglyme and heated in a sealed tube at 150 ℃ for 1 hour. The reaction was filtered, the residue was washed with methanol, and the filtrate was purified by silica gel chromatography to give the desired compound (355mg, 75%) as a white solid. ¹H-NMR(500MHz,CD₃OD) delta ppm 8.04(d,1H),7.67-7.79(m,1H),7.14-7.25(m,1H),7.01-7.11(m,1H),6.66-6.77(m,1H),5.96(s,2H),2.56(s, 3H). OH proton exchange.

Compound 84

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, ammonium hydroxide as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The crude product was purified by reverse phase HPLC to give the desired compound (4.2mg, 13.5%) as a white solid.¹H-NMR(500MHz,CD₃OD)δppm 8.12(d,1H),7.65(s,1H),7.23-7.32(m,1H),7.02-7.14(m,2H),6.77-6.86(m,1H),5.90(s,2H),2.55(s,3H)。NH₂And (4) proton exchange.

Compound 85

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The crude product was purified by reverse phase HPLC to give the desired compound (3.5mg, 7.6%) as a white solid.¹H NMR(500MHz,CD₃OD) delta ppm 8.12(d,1H),7.68(s,1H),7.23-7.35(m,1H),7.02-7.15(m,2H),6.93(t,1H),5.91(s,2H),4.16-4.26(m,1H),4.04-4.12(m,1H),2.58(s, 3H). NH, OH and NH₂And (4) proton exchange.

Compound 86

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, glycine as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The resulting crude product was purified by reverse phase HPLC to give the desired compound (1mg, 2.7%) as a white solid. ¹H NMR(500MHz,CD₃OD) δ ppm 8.27(d,1H),7.74(s,1H),7.23-7.35(m,1H),7.01-7.17(m,2H),6.88(t,1H),5.94(s,2H),4.43(s,2H),2.58(s, 3H). NH and COOH protons are exchanged.

Compound 87

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, 5-aminopentanoic acid as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The crude product was purified by reverse phase HPLC to give the desired compound (43mg, 10.6%) as a tan solid.¹H NMR(500MHz,CD₃OD) delta ppm 8.25(d,1H),7.90(s,1H),7.27-7.36(m,1H),7.04-7.16(m,2H),6.96(t,1H),5.97(s,2H),3.80(t,2H),2.62(s,3H),2.43(t,2H),1.68-1.87(m, 4H). NH and COOH protons are exchanged.

Compound 88

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, piperidine-4-sulfonamide as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The crude product was purified by reverse phase HPLC to give the desired compound (19mg, 42%) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.32(d,1H),7.87(s,1H),7.31(q,1H),7.02-7.16(m,2H),6.92(t,1H),5.96(s,2H),5.02(d,2H),3.36-3.45(m,3H),2.61(s,3H),2.37(d,2H),1.88-2.02(m,2H).NH₂And (4) proton exchange.

Compound 89

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, 4-methylpyrrolidine-3-carboxylic acid as the amine reactant and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The crude product was purified by reverse phase HPLC to give the desired compound (10mg, 24%) as a yellow solid. ¹H NMR(500MHz,CD₃OD) delta ppm 8.32(d,1H),7.88(s,1H),7.27-7.40(m,1H),7.05-7.20(m,2H),6.90-7.03(m,1H),5.99(s,2H),4.32-4.48(m,2H),4.07-4.30(m,1H),3.53-3.74(m,1H),2.83-3.08(m,1H),2.67-2.79(m,1H),2.63(s,3H),1.23-1.40(m, 3H). COOH proton exchange.

Compound 90

The title compound was prepared according to general procedure B, intermediate-1B (1 eq) was used instead of intermediate-1A, 1- ((methylamino) methyl) cyclopentanecarboxylic acid (HBr salt) was used as the amine reactant, and a dioxane solution of the reactant was heated at 90 ℃ for 65 hours. The crude product was purified by reverse phase HPLC to afford the desired compound as a brown solid (9.5mg, 21%).¹H NMR(500MHz,CD₃OD) δ ppm 8.31(d,1H),7.80(s,1H),7.27-7.36(m,1H),7.02-7.16(m,2H),6.90-7.00(m,1H),5.96(s,2H),4.30(s,2H),3.53(d,3H),2.60(s,3H),2.18-2.35(m,2H),1.74-1.86(m,2H),1.64-1.74(m,4H). COOH.

Compound 91

The title compound was prepared according to general procedure B by using intermediate-1B (1 equivalent) instead of intermediate-1A, dimethylamine (60 equivalents) as the amine reactant and no triethylamine and heating a dioxane solution of the reactant at 90 ℃ for 2 hours. The reaction mixture was cooled to 23 ℃, diluted with dichloromethane and washed successively with 1N HCl solution, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the desired compound (13mg, 80%) as a brown solid. ¹H NMR(500MHz,DMSO-d6)δppm 8.22(d,1H),7.69(s,1H),7.28-7.38(m,1H),7.18-7.26(m,1H),7.11(t,1H),6.81(t,1H),5.81(s,2H),3.24(d,6H),2.57(s,3H)。

Compound 92

The title compound was prepared according to general procedure B by using intermediate-1B (1 equivalent) instead of intermediate-1A, pyrrolidine (60 equivalents) as the amine reactant and without triethylamine and heating a dioxane solution of the reactant at 90 ℃ for 2 hours. The reaction mixture was cooled to 23 ℃, diluted with dichloromethane and washed successively with 1N HCl solution, water and brine. The organic layer was then dried over sodium sulfateFiltered and concentrated in vacuo and purified by reverse phase HPLC to give the desired compound (9mg, 49%) as a brown solid.¹H NMR(500MHz,CD₃OD)δppm 8.25(d,1H),7.83(s,1H),7.25-7.41(m,1H),7.02-7.18(m,2H),6.90-7.00(m,1H),4.02(d,4H),5.97(s,2H),2.61(s,3H),1.99-2.24(m,4H)。

Compound 93

The title compound was prepared according to general procedure B by using intermediate-1B (1 eq) instead of intermediate-1A, piperidine (60 eq) as the amine reactant and without triethylamine, and heating a dioxane solution of the reactant at 90 ℃ for 2 hours. The reaction mixture was cooled to 23 ℃, diluted with dichloromethane and washed successively with 1N HCl solution, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the desired compound (0.012g, 63%) as a tan solid.¹H NMR(500MHz,DMSO-d6)δppm 8.26(d,1H),7.69(s,1H),7.33(q,1H),7.19-7.27(m,1H),7.11(t,1H),6.80(t,1H),5.82(s,2H),3.72-3.83(m,4H),2.92-3.06(m,2H),2.57(s,3H),1.65-1.70(m,2H),1.52-1.57(m,1H),1.42-1.50(m,1H)。

Compound 94

The title compound was prepared according to general procedure B by using intermediate-1B (1 equivalent) instead of intermediate-1A, piperazine (60 equivalents) as the amine reactant and no triethylamine and heating a dioxane solution of the reactant at 90 ℃ for 2 hours. The reaction mixture was cooled to 23 ℃, diluted with dichloromethane and washed successively with 1N HCl solution, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the desired compound (10mg, 55%) as a brown solid. ¹H NMR(500MHz,DMSO-d6)δppm 8.27(d,1H),7.70(s,1H),7.33(q,1H),7.16-7.26(m,1H),7.10(t,1H),6.79(t,1H),5.82(s,2H),3.67-3.76(m,4H),2.75-286(m,4H),2.57(s, 3H). NH proton exchange.

Compound 95

The title compound was prepared according to general procedure B by using intermediate-1B (1 eq) instead of intermediate-1A, morpholine (60 eq) as the amine reactant and without triethylamine and heating a dioxane solution of the reactant at 90 ℃ for 2 hours. The reaction mixture was cooled to 23 ℃, diluted with dichloromethane and washed successively with 1N HCl solution, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the desired compound (13mg, 72%) as a brown solid.¹H NMR(500MHz,DMSO-d6)δppm 8.33(d,1H),7.73(s,1H),7.33(q,1H),7.16-7.27(m,1H),7.10(t,1H),6.79(t,1H),5.82(s,2H),3.77-3.85(m,4H),3.69-3.75(m,4H),2.57(s,3H)。

Compound 96

The title compound was prepared according to general procedure B using intermediate-1B (1 equivalent) instead of intermediate-1A, 3,3, 3-trifluoropropane-1-sulfonamide (4 equivalents) as amine reactant, potassium carbonate (4 equivalents) instead of triethylamine, and a DMSO solution of the reactants was heated by microwave at 150 ℃ for 15 minutes. The reaction mixture was cooled to 23 ℃, filtered and purified by reverse phase HPLC to give the desired compound (0.004g, 9.7%) as a tan solid.¹H NMR(500MHz,CD₃OD) delta ppm8.40-8.47(m,1H),7.70(s,1H),7.25-7.34(m,1H),7.03-7.15(m,2H),6.89(t,1H),5.91(s,2H),3.93-4.02(m,2H),2.79-2.92(m,2H),2.57(s,3H). NH proton exchange.

Compound 97

According to aGeneral procedure B the title compound was prepared using intermediate-1B (1 eq) instead of intermediate-1A, propane-1-sulfonamide (4 eq) as the amine reactant, potassium carbonate (4 eq) instead of triethylamine, and the DMSO solution of the reactant was heated by microwave at 150 ℃ for 15 minutes. The reaction mixture was cooled to 23 ℃, filtered and purified by reverse phase HPLC to give the desired compound (5mg, 13.7%) as a brown solid.¹H NMR(500MHz,CD₃OD) delta ppm 8.44-8.54(m,1H),7.63-7.73(m,1H),7.26-7.35(m,1H),7.04-7.14(m,2H),6.91(t,1H),5.92(s,2H),3.69-3.78(m,2H),2.58(s,3H),1.85-1.96(m,2H),1.08(t,3H). NH proton exchange.

Compound 98

The title compound was prepared according to general procedure B using intermediate-1B (1 eq) instead of intermediate-1A, benzenesulfonamide (4 eq) as the amine reactant and potassium carbonate (4 eq) instead of triethylamine, and the reactant solution in DMSO was heated by microwaves at 150 ℃ for 15 minutes. The reaction mixture was cooled to 23 ℃, filtered and purified by reverse phase HPLC to give the desired compound (4mg, 9.6%) as a brown solid.¹H NMR(500MHz,CD₃OD) delta ppm 8.36-8.46(m,1H),8.27(d,2H),7.58-7.63(m,1H),7.45-7.53(m,3H),7.29-7.36(m,1H),7.06-7.18(m,2H),6.90(t,1H),5.92-5.99(m,2H),2.59-2.65(m, 3H). NH proton exchange.

Compound 99

DAST (2.2 equiv.) was added to a solution of intermediate-6 (1 equiv.) in dichloromethane at 0 ℃ and allowed to warm to 23 ℃ over 2 hours. The reaction was then diluted with 7M NH3 in methanol and stirred for 30 minutes. The reaction mixture was concentrated, and the residue was diluted with methanol and filtered to give the desired compound (11mg, 47%) as a white solid.¹H NMR(500MHz,DMSO-d6)δppm8.24(d,1H),7.69(s,1H),7.27-7.41(m,1H),7.15-7.27(m,2H),7.11(t,1H),6.80-6.96(m,2H),5.81(s,2H),4.00(s,2H),3.24(d,3H),2.57(s,3H),1.01-1.06(m,2H),0.83-0.89(m,2H)。

Compound 28

The title compound was prepared according to general procedure B using 5- (aminomethyl) isoxazol-3-ol as the amine reactant and heating the reaction at 100 ℃ for 16 h. The reaction was cooled to room temperature, diluted with water and acidified with 1N HCl solution to pH 3. The resulting precipitate was filtered and dried in vacuo to give the desired compound (68mg, 94% yield, 1:1 dichloromethane solvate) as a white solid.¹H-NMR(500MHz,DMSO-d₆) δ ppm 11.2(s,1H),9.10(d,1H),8.34(br.t,1H),8.27(d,1H),7.51(s,1H),7.33(m,1H),7.24-7.18(m,2H),7.10(app.t,1H),6.87(app.t,1H),6.00(s,1H),5.89(s,2H),4.68(d,2H),3.57(s,8H, dioxane).

Compound 26

The title compound was prepared according to general procedure B using intermediate-1E (described in patent application publication WO 2014/047325) instead of intermediate-1A, using 2- (aminomethyl) -1,1,1,3,3, 3-hexafluoropropan-2-ol as the amine reactant and heating the reaction at 100 ℃ for 15 hours. The reaction was cooled to 23 ℃, diluted with water, acidified to pH 4 with 1N HCl solution and extracted with dichloromethane. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 15-30% ethyl acetate in hexanes to give the desired compound (45mg, 66% yield) as a white solid. ¹H-NMR(500MHz,DMSO-d₆)δppm 9.07(s,1H),8.97(d,1H),8.34(d,1H),8.29(br.t,1H),8.22(s,1H),7.27-7.21(m,2H),7.14(m,1H),7.06(m,1H),7.04(d,1H),4.32(s,2H),4.13(d,2H)。

Compound 30

The title compound was prepared according to general procedure B by substituting intermediate-1E for intermediate-1A and 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide as the amine reactant and heating the reaction at 100 ℃ for 16 h. The reaction was cooled to 23 ℃, diluted with water, acidified to pH 4 with 1N HCl solution and extracted with dichloromethane. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 5-15% acetonitrile-methanol (7:1) in dichloromethane to give the desired compound (47mg, 67% yield) as a white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.14(s,1H),8.96(d,1H),8.28(d,1H),8.01(br.t,1H),7.76(br.s,1H),7.61(br.s,1H),7.25(s,1H),7.27-7.19(m,2H),7.15(m,1H),7.06(app.t,1H),7.03(d,1H),4.34(s,2H),4.11(dd,1H),3.89(dd,1H)。

Compound 102

The target compound is synthesized by two steps:

step 1: synthesis of intermediate-1F

A suspension of phosphorus oxychloride (67 eq) in 5-fluoro-2- (1- (2-fluorobenzyl) -5- (oxazol-2-yl) -1H-pyrazol-3-yl) -pyrimidin-4-ol (intermediate-5F, described in patent application publication WO2012/3405A 1) was heated at 65 ℃ for 2 hours. The reaction mixture was cooled to 23 ℃, dried under a stream of nitrogen, and then concentrated twice from toluene. The resulting pale yellow solid was dried in vacuo and used in the next step without further treatment.

Step 2: synthesis of Compound 102

The title compound was prepared according to general procedure B using intermediate-1F instead of intermediate-1A, 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide as the amine reactant and the reaction was heated at 100 ℃ for 2 days. The reaction was cooled to 23 ℃, diluted with water, and then acidified with 1N HCl solution to pH 6. The resulting precipitate was filtered and dried in vacuo to give the desired compound (59mg, 91% yield) as an off-white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 8.32(m,2H),7.84(br.t,1H),7.75(br.s,1H),7.72(s,1H),7.62(br.s,1H),7.49(s,1H),7.42(s,1H),7.34(m,1H),7.21(m,1H),7.11(app.t,1H),7.02(app.t,1H),6.04(s,2H),4.01(m,2H)。

Compound 103

The title compound was prepared according to general procedure B using intermediate-1F instead of intermediate-1A, 2- (aminomethyl) -1,1,1,3,3, 3-hexafluoropropan-2-ol as amine reactant and heating the reaction at 90-100 ℃ for 5 days. The contents were cooled to 23 ℃, diluted with water, acidified to pH 4 with 1N HCl solution and extracted with dichloromethane. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 15-50% ethyl acetate in hexanes to give the desired compound (38mg, 55% yield) as a white solid.¹H-NMR(500MHz,CDCl₃) δ ppm 8.27(m,2H),7.73(s,1H),7.45(s,1H),7.23(m,1H),7.14(app.t,1H),7.05-7.00(m,2H),6.10(s,2H),5.72(br.s,1H),4.15(d, 2H). No exchangeable OH protons were observed.

Compound 109

Acetic acid (5.5 equiv.) was added to a suspension of intermediate-1A (1 equiv.) and zinc dust (3.4 equiv.) in THF and the reaction was heated at 75 ℃ for 72 h. After the reaction is finishedAfter completion, the reaction was quenched by addition of 1N HCl solution and extracted with ethyl acetate. The organic layer was dried and concentrated to give the desired compound (47mg, yield 82%) as a brown solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.05-9.18(m,1H),8.96(s,2H),7.60-7.71(m,1H),7.31-7.38(m,1H),7.28(d,1H),7.19-7.26(m,1H),7.12(t,1H),6.91-6.99(m,1H),5.93(s,2H)。

Compound 37

Compound 109(1.0 equiv.), morpholine (7.0 equiv.) in anhydrous DMSO was heated to 120 ℃ for 18 hours. The contents were cooled to 23 ℃, diluted with water and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 30-50% acetonitrile-methanol (7:1) in dichloromethane to give the desired compound (34mg, 57% yield) as an off-white solid.¹H-NMR(500MHz,DMSO-d₆)δppm9.09(d,1H),8.54(s,2H),7.55(s,1H),7.34(m,1H),7.27(d,1H),7.22(m,1H),7.11(app.t,1H),6.90(app.t,1H),5.89(s,2H),3.77(m,4H),3.29(m,4H)。

Compound 8

A solution of compound 109(1.0 equiv), 2-aminoethanol (7.0 equiv) in anhydrous DMSO was heated to 120 ℃ for 22 hours. The reaction was cooled to 23 ℃, diluted with water and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 25-50% acetonitrile-methanol (7:1) in dichloromethane to give the desired compound (36mg, 65% yield) as a pale yellow solid. ¹H-NMR(500MHz,DMSO-d₆)δppm 9.08(d,1H),8.21(s,2H),7.46(s,1H),7.33(m,1H),7.25(d,1H),7.22(m,1H),7.11(app.t,1H),6.88(app.t,1H),6.27(t,1H),5.87(s,2H),4.80(t,1H),3.59(dt,2H),3.21(dt,2H)。

Compound 9

A solution of compound 109(1.0 equiv), ethane-1, 2-diamine (7.0 equiv) in anhydrous DMSO was heated to 120 ℃ for 8 hours. The reaction was cooled to 23 ℃, poured into saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by preparative HPLC eluting with a gradient of 30-80% acetonitrile/water (containing 0.1% TFA) to give the desired compound (33mg, yield 51%, TFA salt) as a pale yellow solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.10(d,1H),8.24(s,2H),7.79(br.s,2H),7.47(s,1H),7.34(m,1H),7.25(d,1H),7.22(m,1H),7.11(app.t,1H),6.90(app.t,1H),6.37(br.s,1H),5.88(s,2H),3.40(m,2H),3.01(m,2H)。

Compound 6

An anhydrous DMSO suspension of compound 109(1.0 eq), glycinamide hydrochloride (7.0 eq) and sodium bicarbonate (7.0 eq) was heated at 120-130 ℃ for 2 days. Glycinamide hydrochloride (7.0 equiv.) and sodium bicarbonate (7.0 equiv.) were added further and the reaction was heated at 130 ℃ for an additional 2 days. The reaction was cooled to 23 ℃, poured into half-saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 20-60% acetonitrile-methanol (7:1) in dichloromethane followed by purification using reverse phase HPLC eluting with a gradient of 30-80% acetonitrile/water (with 0.1% TFA) to give the desired compound (2.0mg, 3.4% yield) as a white solid. ¹H-NMR(500MHz,CD₃OD)δppm 8.77(d,1H),8.26(s,2H),7.41(s,1H),7.27(m,1H),7.09(m,1H),7.03(app.t,1H),6.89(d,1H),6.87(app.t,1H),5.96(s,2H),3.94(s,2H)。

Compound 21

Compound 109(1.0 equiv.), 3-aminopropane-1, 2-diol (7.0 equiv.) in anhydrous DMSO was heated to 120 ℃ for 18 hours. The reaction was cooled to 23 ℃, diluted with water and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 30-50% acetonitrile-methanol (7:1) in dichloromethane to give the desired compound (34mg, 57% yield) as an off-white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.08(d,1H),8.22(s,2H),7.46(s,1H),7.33(m,1H),7.24(d,1H),7.22(m,1H),7.11(app.t,1H),6.88(app.t,1H),6.22(t,1H),5.87(s,2H),4.91(d,1H),4.68(t,1H),3.65(m,1H),3.39(m,2H),3.27(m,1H),3.03(m,1H)。

Compound 22

A solution of compound 109(1.0 equiv.), 2- (methylsulfonyl) ethylamine hydrochloride (7.0 equiv.) and triethylamine (7.0 equiv.) in anhydrous DMSO was heated at 120 ℃ for 4 days. An additional amount of 2- (methylsulfonyl) ethylamine hydrochloride (7.0 equiv.) and triethylamine (7.0 equiv.) were added and the reaction heated at 120 ℃ for an additional 7 days. The reaction was cooled to 23 ℃, diluted with water and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 70-100% ethyl acetate in hexanes to give the desired compound (23mg, 33% yield) as an off-white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.08(d,1H),8.24(s,2H),7.49(s,1H),7.34(m,1H),7.25(d,1H),7.22(m,1H),7.11(app.t,1H),6.88(app.t,1H),6.47(t,1H),5.88(s,2H),3.61(dt,2H),3.41(t,2H),3.05(s,3H)。

Compound 24

A solution of compound 9(1.0 equiv., TFA salt) in dichloromethane was added triethylamine (5.0 equiv.) and methanesulfonyl chloride (1.1 equiv.) at 0 deg.C. The reaction mixture was heated to 23 ℃ and stirred at this temperature for 3 hours. The reaction was poured into half-saturated sodium bicarbonate solution and extracted with dichloromethane and ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with 25% acetonitrile-methanol (7:1) in dichloromethane to give the desired compound (9.5mg, 75% yield) as a pale yellow solid.¹H-NMR(500MHz,CDCl₃) δ ppm 8.45(d,1H),8.14(s,2H),7.30(s,1H),7.18(m,1H),7.01(m,1H),6.95(app.t,1H),6.84(app.t,1H),6.60(d,1H),5.97(s,2H),5.53(br.s,1H),3.43-3.35(m,4H),2.98(s,3H). no exchangeable sulfonamide NH protons were observed.

Compound 7

To a solution of intermediate-13 (1.0 eq) in anhydrous dioxane was added 2-aminoethanol (4.0 eq). The reaction mixture turned into an orange suspension. After 20 h, water was added, the solid was filtered and dried in vacuo to give the desired compound (160mg, 89% yield) as a tan solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.19(s,1H),9.12(d,1H),8.83(t,1H),7.73(s,1H),7.34(m,1H),7.28(d,1H),7.23(m,1H),7.11(app.t,1H),6.87(app.t,1H),5.95(s,2H),4.96(t,1H),3.80(dt,2H),3.67(dt,2H)。

Compound 82

To a suspension of compound 7 in MeOH/ethyl acetate (1:1) under nitrogen, 10% palladium on carbon (0.2 equiv.) was added. The resulting mixture was stirred for 1 hour and 40 minutes under hydrogen (using a balloon). The reaction vessel was then flushed with nitrogen and the contents filtered through celite. The solvent was removed in vacuo to give the desired intermediate, 2- ((5-amino-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) ethanol (compound 82), which was used in the next step without further treatment.

Compound 10

Triethylamine (2.0 equivalents) was added to a suspension of 2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidine-4, 5-diamine (WO 2012/3405A1, WO2013/101830A1 described in the previous patent) (1.0 equivalents) in anhydrous THF, followed by 1,3, 2-dioxathiolane-2, 2(1.2 equivalents). After 18 hours, an additional amount of 1,3, 2-dioxathiolane-2, 2-dioxide (0.30 equivalents) was added and the reaction was stirred for 5 hours. The reaction mixture was then concentrated in vacuo, resuspended in 6N HCl in THF/water (3:1v/v) and heated at 60 ℃ for 18 h. After cooling to 23 ℃ the reaction was poured into half-saturated sodium bicarbonate solution and extracted with dichloromethane/iPrOH (4: 1). The organic layer was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with 70-100% acetonitrile-methanol (7:1) in dichloromethane to give the desired compound (31mg, 62% yield) as an off-white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.19 (d,1H),8.83(br.s,1H),8.10(br.s,1H),7.58(s,1H),7.46(s,1H),7.39(m,1H),7.26(d,1H),7.24(m,1H),7.19-7.10(m,2H),5.99(s,1H),5.89(s,2H),5.07(t,1H),4.50(m,2H),3.62(m,2H)。

Compound 60

To a suspension of intermediate-14 (1.0 equiv.) in dichloromethane was added DA in one portion at 0 deg.CST (2.2 equiv.). The mixture was heated to 23 ℃ and stirred for 24 hours. The solvent was removed in vacuo and the residue was dissolved in ammonium hydroxide (concentrated), heated at 100 ℃ for 24 h and the solvent removed under nitrogen. The crude product was purified by reverse phase HPLC to give the desired compound (1.6mg, 3% yield) as a white solid. ¹H-NMR(500MHz,CD₃OD)δppm 8.83(m,1H),8.32(m,1H),7.67(m,1H),7.32(m,1H),7.11(m,2H),6.98(m,2H),6.04(m,2H),4.96(m,2H),3.47(m,2H),2.75(m,1H),2.09(m,2H),1.91(m,2H)。

Compound 105

The compound is prepared by 5 steps:

step 1: synthesis of 3- (ethoxycarbonyl) -1- (2-fluorobenzyl) -1H-pyrazole-5-carboxylic acid

To a suspension of diethyl 1- (2-fluorobenzyl) -1H-pyrazole-3, 5-dicarboxylate (previously described in the literature) (1 eq) in ethanol was slowly added potassium hydroxide over a period of 1.5 hours, since not all starting materials were completely brought into solution. After stirring at 23 ℃ for 15 h, LCMS indicated the starting material was still present. An additional 20 mol% potassium hydroxide was added and stirring continued at 23 ℃ for 1.5 hours, followed by an additional 30 mol% potassium hydroxide and stirring for an additional 2 hours. The solution was poured into saturated NH4Cl solution and extracted with dichloromethane (6 ×). The combined organic phases were dried over magnesium sulfate, filtered and the solvent was removed in vacuo to give the desired intermediate 3- (ethoxycarbonyl) -1- (2-fluorobenzyl) -1H-pyrazole-5-carboxylic acid (2.98g, 108% yield) as a white solid. The crude product was used in the next step without further purification.

Step 2: synthesis of 1- (2-fluorobenzyl) -5- (hydroxymethyl) -1H-pyrazole-3-carboxylic acid

To 3- (ethoxycarbonyl) -1- (2-fluorobenzyl) -1H-pyrazole-5-carboxylic acid (1 am) at 0 deg.CAmount) to a solution of 10M borane-methyl sulfide complex (3 equivalents). After the evolution of gas had ceased (15 minutes), the solution was slowly heated to 23 ℃ and then stirred at 65 ℃ for 4 hours. The reaction was cooled to 23 ℃, quenched with 1N HCl (aq) and stirred for 1 hour. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give the desired intermediate, (2-fluorobenzyl) -5- (hydroxymethyl) -1H-pyrazole-3-carboxylic acid (0.59g, 74% yield) as a colourless oil. ¹H-NMR(500MHz,DMSO-d₆)δppm 12.67(m,1H),7.37(m,1H),7.24(m,1H),7.16(m,1H),7.03(m,1H),6.65(m,1H),5.46(s,2H),4.52(m,2H)。

Step 3: synthesis of methyl 1- (2-fluorobenzyl) -5- (methoxymethyl) -1H-pyrazole-3-carboxylate

To a solution of 1- (2-fluorobenzyl) -5- (hydroxymethyl) -1H-pyrazole-3-carboxylic acid (1 eq) in DMF was added sodium hydride (2.1 eq) at 0 ℃. The solution was stirred at 0 ℃ for 30 minutes and at 23 ℃ for 30 minutes. To this solution was added methyl iodide (4.2 equiv.) and stirred for 18 hours. The mixture was diluted with ethyl acetate and washed with water. The organic layer was dried, filtered and evaporated to give a crude product as an oil which was purified by silica gel chromatography to give the desired intermediate methyl (2-fluorobenzyl) -5- (methoxymethyl) -1H-pyrazole-3-carboxylate (260mg, 42% yield) as a clear colorless oil.¹H-NMR(500MHz,DMSO-d₆)δppm 7.34(m,1H),7.22(m,1H),7.13(m,1H),6.90(m,2H),5.76(m,2H),4.37(s,2H),3.81(m,3H),3.25(s,3H)。

Step 4: synthesis of 1- (2-fluorobenzyl) -5- (methoxymethyl) -1H-pyrazole-3-carboxamidine (intermediate-19)

To a toluene suspension of ammonia hydrochloride (5.3 equivalents) at 0 ℃ was added trimethylaluminum 2MToluene solution (5.3 eq). The mixture was removed from the ice bath and stirred at 23 ℃ until bubbling ceased. To the mixture was added a solution of 1- (2-fluorobenzyl) -5- (methoxymethyl) -1H-pyrazole-3-carboxylic acid methyl ester (1 eq) in toluene and stirred at 80 ℃ for 24 hours. The mixture was cooled in an ice bath and quenched slowly with methanol and the resulting white precipitate was removed by filtration through celite. The filtrate was concentrated and dried in vacuo to give the desired intermediate, 1- (2-fluorobenzyl) -5- (methoxymethyl) -1H-pyrazole-3-carboxamidine (258mg, yield 100%) as an off-white solid. ¹H-NMR(500MHz,DMSO-d₆)δppm 7.29(m,6H),6.85(m,1H),5.55(s,2H),4.36(s,2H),3.34(s,1H),3.26(s,3H)。

Step 5: synthesis of Compound 105

The ethanol mixture containing 1- (2-fluorobenzyl) -5- (methoxymethyl) -1H-pyrazole-3-carboxamidine (intermediate-19, 4 equivalents) was stirred at 100 ℃ for 1 hour. The mixture was cooled and the solvent was removed in vacuo. The crude product was purified by reverse phase chromatography to give the desired compound (11mg, 20% yield) as an off-white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 8.18(m,1H),7.31(d,1H),7.18(t,1H),7.10(m,2H),6.93(t,1H),5.87(s,2H),4.37(s,2H),3.27(s,3H)。

Compound 20

A solution of compound 105(1 equivalent) in phosphorus trichloride (excess) was heated at 65 ℃ for 2 hours. The solution was cooled and the solvent was removed under nitrogen. The resulting residue was dissolved in dioxane and water (3:1), and 2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide (10 equivalents) and triethylamine (20 equivalents) were added. The solution was stirred at 100 ℃ for 18 hours. The solvent was evaporated and the crude product was purified by reverse phase chromatography to give the desired compound (6.7mg, 47% yield).¹H-NMR(500MHz,CDCl₃)δ8.16(m,1H),7.22(m,1H),7.13(m,1H),7.05(s,3H),6.80(m,1H),6.29(m,1H),6.08(d,2H),4.58(s,2H),4.19(m,2H),3.47(m,3H)。

Compound 5

The target compound is synthesized by 2 steps:

step 1: synthesis of 2- (((benzyloxy) carbonyl) amino) -2-methylpropanoic acid

A solution of water and 1, 4-dioxane (2:1) containing sodium carbonate (3 equivalents), 2-amino-2-methylpropionic acid (1.0 equivalent) and benzyl chloroformate (1.1 equivalents) was stirred at 23 ℃ for 24 hours. The mixture was diluted with ethyl acetate and washed with 1N HCl solution. The organic layer was dried, filtered and evaporated to give the desired intermediate, 2- (((benzyloxy) carbonyl) amino) -2-methylpropanoic acid (825mg, 72% yield) as a clear oil. ¹H NMR(500MHz,CD₃OD)δppm 7.32-7.38(m,5H),5.03-5.09(m,2H),1.43-1.50(m,6H)。

Step 2: synthesis of Compound 5

The title compound was prepared according to general procedure C using 2- (((benzyloxy) carbonyl) amino) -2-methylpropanoic acid (1 eq) as the acid reactant, 2.5 eq of T3P, heating the reaction at 70 ℃ for 24 h, and extraction with ethyl acetate in workup. The crude product was purified by silica gel chromatography to give the desired compound (87mg, 7% yield) as a brown solid.¹H NMR(500MHz,CD₃OD)δppm 8.79(s,1H),8.68(d,1H),8.09-8.20(m,1H),7.98(s,1H),7.52(s,1H),7.21-7.39(m,4H),7.17(br.s.,1H),7.02-7.15(m,2H),6.85-6.92(m,2H),5.99(s,2H),5.06(s,2H),1.48-1.51(m,6H)。

Compound 33

An ethanol solution containing palladium on carbon (0.1 eq) and compound 5(1 eq)) was hydrogenated at ambient temperature under hydrogen for 24 hours. The mixture was filtered through an Acro disc, and the filtrate was concentrated in vacuo to give the desired compound (66mg, yield 99%) as a white solid.¹H NMR(500MHz,CDCl₃)δppm 8.68-8.74(m,1H),8.46(s,1H),8.14-8.18(m,1H),7.44-7.48(m,1H),7.19(q,1H),7.00-7.09(m,1H),6.96(t,1H),6.81(t,1H),6.58-6.63(m,1H),6.02(s,2H),1.44-1.48(m,6H)。

Compound 46

The target compound is synthesized by 2 steps:

step 1: synthesis of 2- (((benzyloxy) carbonyl) amino) -2-methylbutyric acid

A solution of 2-amino-2-methylbutyrate hydrochloride (1 equivalent), sodium carbonate (3 equivalents) and benzyl chloroformate (1.1 equivalents) in 1, 4-dioxane and water (2:1) was stirred at 23 ℃ for 24 h. The mixture was diluted with ethyl acetate and washed with 1N HCl solution. The organic layer was dried, filtered and evaporated to give the desired intermediate, 2- (((benzyloxy) carbonyl) amino) -2-methylbutyric acid (499mg, 99% yield) as a clear oil. ¹H NMR(500MHz,CD₃OD)δppm 7.30-7.38(m,5H),5.01-5.09(m,2H),1.85-1.95(m,2H),1.43-1.50(m,3H),0.86(t,3H)。

Step 2: synthesis of Compound 46

The title compound was prepared according to general procedure C, using 2- ((((benzyloxy) carbonyl) amino) -2-methylbutyric acid (1 eq) as acid reactant, 2.5 eq of T3P, heating the reaction at 70 ℃ for 3 days, and using ethyl acetate in workupAnd (4) ester extraction. The crude product was purified by silica gel chromatography (gradient elution from 0 to 100% ethyl acetate in hexanes) to afford the desired compound (40mg, 5% yield).¹H NMR(500MHz,CD₃OD)δppm 8.78(s,1H),8.67(d,1H),8.12(br.s.,1H),7.51(s,1H),7.22-7.36(m,4H),7.17(d,1H),7.06-7.13(m,1H),7.04(t,1H),6.82-6.96(m,3H),5.98(s,2H),5.05(br.s.,2H),1.84-2.04(m,2H),1.46-1.51(m,3H),0.85-0.94(m,3H)。

Compound 47

A solution containing palladium on carbon (0.1 eq) and compound 46(1 eq) was hydrogenated at 23 ℃ for 24 hours. The mixture was filtered through an Acro disk, and the filtrate was concentrated in vacuo to give the desired compound (25mg, yield 100%) as a clear oil.¹H NMR(500MHz,CDCl₃)δppm 8.71(d,1H),8.46(s,1H),8.18(d,1H),7.47(s,1H),7.17-7.24(m,1H),7.04(d,1H),6.97(t,1H),6.82(t,1H),6.61(s,1H),6.03(s,2H),1.91-2.02(m,2H),1.44(s,3H),0.92-0.97(m,3H)。

Compound 55

The title compound was prepared according to general procedure B, using 2-methylbutane-1, 2-diamine (1.1 equivalents) as the amine reactant, 1 equivalent of triethylamine and a DMF solution of the reactant was stirred at 23 ℃ until complete consumption of starting material was monitored by LC/MS. The reaction was diluted with ethyl acetate and water. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (gradient elution from 0-10% methanol in dichloromethane) to give the desired compound (67mg, yield 15%) as a white solid. ¹H NMR(500MHz,CD₃OD)δppm 8.67-8.74(m,1H),8.02(d,1H),7.38(s,1H),7.21(q,1H),7.01-7.08(m,1H),6.97(t,1H),6.84(s,1H),6.77(t,1H),5.87-5.94(m,2H),3.25-3.29(m,2H),1.45-1.55(m,2H),1.07-1.12(m,3H),0.90-0.97(m,3H)。

Compound 67

The title compound was prepared according to general procedure B using 2-cyclopropylpropane-1, 2-diamine dihydrochloride (2 equivalents) as the amine reactant and 4 equivalents of triethylamine and the DMF solution of the reactant was stirred at 23 ℃ until complete consumption of starting material as monitored by LC/MS. The reaction was diluted with ethyl acetate and water. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (gradient elution from 0 to 10% methanol in dichloromethane) to give the desired compound (81mg, yield 67%) as a clear oil.¹H NMR(500MHz,CD₃OD)δppm 8.75(s,1H),8.08(d,1H),7.44(s,1H),7.21-7.30(m,1H),7.08(t,1H),7.01(t,1H),6.89(s,1H),6.79(t,1H),5.95(s,2H),3.35(s,2H),1.03-1.09(m,1H),1.02(s,3H),0.37-0.51(m,4H)。

Compound 68

The title compound was prepared according to general procedure B using 1- (aminomethyl) cyclopropylamine (as 2HCl salt, 2 equivalents) as the amine reactant, 8 equivalents of triethylamine and a solution of the reactant in DMF was stirred at 23 ℃ until complete consumption of starting material was monitored by LC/MS. The reaction was diluted with ethyl acetate and water. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (gradient elution from 0 to 10% methanol in dichloromethane) to give the desired compound (54mg, 40% yield) as a white solid.¹H NMR(500MHz,CD₃OD)δppm 8.75(s,1H),8.05(d,1H),7.42(s,1H),7.17-7.33(m,1H),7.05-7.15(m,1H),7.02(t,1H),6.89(s,1H),6.81(t,1H),5.95(s,2H),3.69(s,2H),0.59-0.77(m,4H)。

Compound 106

The title compound was prepared according to general procedure B using (R) -3,3, 3-trifluoro-2-methylpropane-1, 2-diamine dihydrochloride as amine reactant and 6 equivalents of triethylamine and stirring DMF solution of the reactant at 23 ℃ until complete consumption of starting material was monitored by LC/MS. The solution was diluted with ethyl acetate and water. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (gradient elution from 0 to 10% methanol in dichloromethane) to give the desired compound (108mg, 84% yield) as a white solid. ¹H NMR(500MHz,CD₃OD)δppm 8.76(d,1H),8.11(d,1H),7.41(s,1H),7.19-7.36(m,1H),7.06-7.13(m,1H),7.03(t,1H),6.89(d,1H),6.85(t,1H),5.95(s,2H),3.80-3.98(m,2H),1.32(s,3H)。

Compound 107

The title compound was prepared according to general procedure B using (R) -2- (((S) -3-amino-1, 1, 1-trifluoro-2-methylpropan-2-yl) amino) -2-phenylethanol as amine reactant and 6 equivalents of triethylamine and stirring a DMF solution of the reactant at 23 ℃ until complete consumption of starting material was monitored by LC/MS. The solution was diluted with ethyl acetate and water. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (gradient elution from 0 to 10% methanol in dichloromethane) to give the desired compound (72mg, yield 70%) as a white solid.¹H-NMR(500MHz,CD₃OD)δppm 8.67-8.78(m,1H),8.12(d,1H),7.40(s,1H),7.09-7.33(m,6H),7.01-7.07(m,1H),6.97(t,1H),6.87(d,1H),6.82(t,1H),5.92(s,2H),4.12(dd,1H),3.80-3.99(m,2H),3.48(dd,1H),3.31(d,1H),1.10(s,3H)。

Compound 108

The title compound was prepared according to general procedure B using (S) -3,3, 3-trifluoro-2-methylpropane-1, 2-diamine dihydrochloride as the amine reactant and 6 equivalents of triethylamine and stirring the DMF solution of the reactant at 23 ℃ until complete consumption of starting material was monitored by LC/MS. The solution was diluted with ethyl acetate and water. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by silica gel chromatography (gradient elution from 0 to 10% methanol in dichloromethane) to give the desired compound (58mg, 46% yield) as a white solid. ¹H-NMR(500MHz,CD₃OD)δppm 8.75(s,1H),8.10(d,1H),7.40(s,1H),7.26(q,1H),7.08(s,1H),7.02(s,1H),6.87(d,1H),6.84(t,1H),5.94(s,2H),3.88-3.99(m,1H),3.80-3.88(m,1H),1.31(s,3H)。

Compound 111

The target compound is prepared by 5 steps:

step 1: synthesis of N-methoxy-N-methylisothiazole-3-carboxamide

To a suspension of isothiazole-3-carboxylic acid (2g, 15.49mmol) in DCM at 0 deg.C was added oxalyl chloride (1.3 eq.) followed by 3 drops of DMF. Bubbles began to appear and 10 minutes later the reaction was warmed to 23 ℃. The mixture was stirred for 3 hours and then cooled to 0 ℃. N, O-dimethylhydroxylamine hydrochloride (1.3 equivalents) was added to the reaction, and triethylamine (3.5 equivalents) was added dropwise via syringe over 10 minutes. The reaction was slowly heated to 23 ℃ overnight and stirred for a total of 15 hours. The reaction mixture was diluted with 1N HCl solution and dichloromethane (1:1 ratio). The layers were separated and the aqueous layer was extracted 2 times with DCM. The combined organic layers were dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (using hexane/ethyl acetate mixture as eluent) to give the desired intermediate, N-methoxy-N-methylisothiazole-3-carboxamide (1g, 38% yield) as a pale yellow solid.¹H NMR(400MHz,CDCl₃)δppm 8.67(d,1H),7.69(br s,1H),3.80(s,3H),3.46(br s,3H)。

Step 2: (E) synthesis of ethyl (E) -4- (isothiazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoate

To a solution of N-methoxy-N-methylisothiazole-3-carboxamide (200mg, 1.161mmol) and ethyl propiolate (1.5 equiv) in THF at-55 deg.C (2:1 ethanol/water/dry ice) was added sodium bis (trimethylsilyl) amide (1.4 equiv., 1N in THF) over 5 minutes. The reaction was allowed to warm to-45 ℃ over 15 minutes, then to 30 ℃ over 15 minutes, and then stirred for an additional 15 minutes. The reaction was treated with 1N aqueous HCl, stirred at-30 ℃ for 3 minutes, and then acidified to pH 2 with 10% aqueous citric acid. The mixture was warmed to 23 ℃ and then partitioned between dichloromethane and water. The layers were separated and the aqueous layer was extracted 2 times with dichloromethane and 1 time with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (using hexane/ethyl acetate mixture as eluent) to give the desired intermediate, ethyl (E) -4- (isothiazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoate (234mg, 75% yield) as an oil. ¹H NMR(400MHz,CDCl₃)δppm 8.58(d,1H),7.78(d,1H),6.43(s,1H),4.42(q,2H),3.70(s,3H),3.16(s,3H),1.35(t,3H)。

Step 3: synthesis of ethyl 1- (2-fluorobenzyl) -5- (isothiazol-3-yl) -1H-pyrazole-3-carboxylate

To a solution of (2-fluorobenzyl) hydrazine hydrochloride (1.1 eq) in ethanol/water (10:1 ratio) was added an aqueous solution of potassium carbonate (0.55 eq) followed immediately by a solution of (E) -4- (isothiazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoic acid ethyl ester (1 eq) in ethanol. Will reactThe material was stirred at 23 ℃ for 3 hours and then diluted with dichloromethane and 1N aqueous HCl (3:1 ratio). The layers were separated and the aqueous layer was extracted 2 times with dichloromethane. The combined organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (using hexane/ethyl acetate mixture as eluent) to give the desired intermediate, ethyl 1- (2-fluorobenzyl) -5- (isothiazol-3-yl) -1H-pyrazole-3-carboxylate (166mg, 57.9% yield) as a white solid.¹H NMR(400MHz,CDCl₃)δppm 8.66(d,1H),7.44(d,1H),7.18(s,1H),7.11-7.17(m,1H),6.90-7.02(m,2H),6.76(td,1H),6.10(s,2H),4.41(q,2H),1.39(t,3H)。

Step 4: synthesis of 1- (2-fluorobenzyl) -5- (isothiazol-3-yl) -1H-pyrazole-3-carboxamidine

To a toluene suspension of ammoniacal hydrochloride (5.5 equivalents) was added a 2M solution of trimethylaluminum (5 equivalents) in toluene. After bubbling stopped, 1- (2-fluorobenzyl) -5- (isothiazol-3-yl) -1H-pyrazole-3-carboxylic acid ethyl ester (1 eq) was added directly to the solution. The reaction mixture was heated to 100 ℃ (with periodic release of pressure as the temperature increased) in a closed vial for 3 hours. The reaction was cooled to 0 ℃ and then methanol (10 equivalents) was added. After stirring at 23 ℃ for 15 minutes, the mixture was diluted with toluene and filtered through celite. The solid was washed with 5mL of methanol, then the filtrate was concentrated in vacuo to give the desired intermediate, 1- (2-fluorobenzyl) -5- (isothiazol-3-yl) -1H-pyrazole-3-carboxamidine (130mg, yield 86%) as a yellow solid. The product was used without further purification. ¹H NMR(400MHz,DMSO-d₆)δppm 9.27(d,1H),7.85(s,1H),7.74(d,1H),7.30-7.37(m,1H),7.19-7.24(m,1H),7.11(td,1H),6.91(td,1H),6.09(s,2H)。

Step 5: synthesis of Compound 111

An ethanol suspension of 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-olate (3 eq) and 1- (2-fluorobenzyl) -5- (isothiazol-3-yl) -1H-pyrazole-3-carboxamidine (1 eq) was stirred at 90 ℃ for 1H 30 min. The solvent was removed in vacuo and the black solid was suspended in dichloromethane. The solid was filtered and then purified by silica gel chromatography (using hexane/ethyl acetate mixture as eluent) to give the desired compound (53mg, yield 33.1%) as a brown solid.¹H NMR(400MHz,CD₃OD)δppm 9.00(d,1H),8.04(d,1H),7.74(d,1H),7.45(s,1H),7.22-7.30(m,1H),6.99-7.12(m,2H),6.89(br t,1H),6.17(s,2H)。

Compound 112

Reacting 8-oxa-2-azaspiro [4.5 ]]Decane (1.5 equiv.), triethylamine (10 equiv.) and a solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equiv.) in dioxane were stirred at 130 ℃ for 3 hours. The crude product was purified by reverse phase HPLC to give the desired compound (15mg, 54.8% yield) as an off-white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.28(d,1H),7.74(s,1H),7.34(q,1H),7.20-7.26(m,1H),7.11(t,1H),6.82(t,1H),5.83(s,2H),3.83(br.s.,2H),3.58-3.66(m,6H),2.58(s,3H),1.91(br.s.,2H),1.57-1.64(m,2H),1.50-1.57(m,2H)。

Compound 113

Reacting 2-oxa-7-azaspiro [3.5 ]]Nonane (1.5 eq), triethylamine (10 eq) and a solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq) in dioxane were stirred at 130 ℃ for 3 hours. The crude product was purified by reverse phase HPLC to give the desired compound (17mg, 64.1% yield) as a white solid. ¹H-NMR(500MHz,DMSO-d₆)δppm 8.28-8.33(m,1H),7.71-7.75(m,1H),7.30-7.37(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.81(t,1H),5.82(s,2H),4.37(s,4H),3.70-3.76(m,4H),2.58(s,3H),1.88-1.93(m,4H)。

Compound 114

A solution of 3, 3-difluoroazetidine (1.5 eq), triethylamine (10 eq) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq) in DMF was stirred at 130 ℃ for 3H. The crude product was purified by reverse phase HPLC to give the desired compound (11mg, 45.0% yield) as a white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 8.41(d,1H),7.72(s,1H),7.30-7.36(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.79(t,1H),5.83(s,2H),4.72(t,4H),2.57(s,3H)。

Compound 115

A solution of 2, 2-dimethylthiomorpholine 1, 1-dioxide (1.5 eq), triethylamine (10 eq) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq) in DMF was stirred at 130 ℃ for 3 hours. The crude product was purified by reverse phase HPLC to give the desired compound (9mg, yield 31.4%) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.42(d,1H),7.76(s,1H),7.31-7.37(m,1H),7.19-7.25(m,1H),7.12(t,1H),6.86(t,1H),5.82(s,2H),4.25(br.s.,2H),4.00(br.s.,2H),3.43(t,2H),2.59(s,3H),1.30(s,6H)。

Compound 116

(3R, 4S) -piperidine-3, 4-diol (1.5 eq.), triethylamine (10 eq.) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazole-5-Acetyl) ethanone (intermediate-1B, 1 eq.) in DMF was stirred at 130 ℃ for 3 hours. The crude product was purified by reverse phase HPLC to give the desired compound (14mg yield, 54.0%) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.29(dd,1H),7.73(d,1H),7.30-7.37(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.81(t,1H),5.83(s,2H),4.06(br.s.,1H),3.86-3.95(m,1H),3.71-3.71(m,1H),3.64-3.80(m,2H),3.51-3.59(m,1H),2.58(s,3H),1.75-1.85(m,1H),1.66(d,1H)。

Compound 117

A solution of 4- (hydroxymethyl) piperidin-4-ol (1.5 equivalents), triethylamine (10 equivalents) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) in DMF was stirred at 130 ℃ for 3 hours. The crude product was purified by reverse phase HPLC to give the desired compound (16mg, 59.8% yield) as a white solid. ¹H NMR(500MHz,DMSO-d₆)δppm 8.30(t,1H),7.74-7.78(m,1H),7.30-7.36(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.81(t,1H),5.83(s,2H),4.38(br.s.,2H),3.41(t,2H),3.22(s,2H),2.58(s,3H),1.68(t,2H),1.49(d,2H)。

Compound 118

2-oxa-6-azaspiro [3.3 ]]Heptane (1.5 equivalents), triethylamine (10 equivalents) and a solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) in DMF were stirred at 130 ℃ for 3H. The crude product was purified by reverse phase HPLC to give the desired compound (8mg, yield 32.2%) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.27(d,1H),7.67(s,1H),7.33(q,1H),7.20-7.26(m,1H),7.11(t,1H),6.79(t,1H),5.83(s,2H),4.73(s,4H),4.47(br.s.,4H),2.57(s,3H)。

Compound 119

A suspension of isothiazolidine 1, 1-dioxide (1.2 equiv.), cesium carbonate (1.5 equiv.) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equiv.) in DMF was stirred at 130 ℃ for 2H. The solution was diluted with ethyl acetate, washed with water and brine. The organics were combined, dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude residue was purified by reverse phase HPLC to give the desired compound (11mg, 42.0% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.80(d,1H),7.78(s,1H),7.31-7.37(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.84(t,1H),5.85(s,2H),4.15(t,2H),3.64(t,2H),2.59(s,3H),2.50(t,2H)。

Compound 120

A solution of 1- (methylsulfonyl) -piperazine (1.5 equivalents), triethylamine (10 equivalents) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) in DMF was stirred at 130 ℃ for 3 hours. The crude product was purified by reverse phase HPLC to give the desired compound (14mg, 48.7% yield) as a white solid. ¹H NMR(500MHz,DMSO-d₆)δppm 8.38(d,1H),7.76(s,1H),7.30-7.37(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.80(t,1H),5.83(s,2H),3.93(br.s.,4H),3.28(d,4H),2.91(s,3H),2.58(s,3H)。

Compound 121

A solution of tert-butyl azetidin-3-ylcarbamate (1.5 equiv.), triethylamine (10 equiv.) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equiv.) in DMF was stirred at 130 ℃ for 1 hour.The solution was diluted with ethyl acetate and washed with 1N aqueous HCl, water and brine. The solution was concentrated in vacuo, redissolved in DCM, and trifluoroacetic anhydride (1 eq) was added and stirred at 23 ℃ for 2 h. The solvent was removed in vacuo and the crude product was purified by reverse phase HPLC to give the desired compound (12mg, 27.6% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 10.11(d,1H)8.31(d,1H)7.69(s,1H)7.30-7.36(m,1H)7.20-7.26(m,1H)7.11(t,1H)6.80(t,1H)5.83(s,2H)4.77(dq,1H)4.59(br.S,2H)4.30(d,2H)2.57(s,3H)。

Compound 122

A solution of N- (azetidin-3-yl) -1-hydroxycyclopropanecarboxamide (1.5 eq), triethylamine (10 eq) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq) in DMF was stirred at 130 ℃ for 2H. The crude product was purified by reverse phase HPLC to give the desired compound (11mg, 39% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.75(d,1H),8.28(d,1H),7.69(s,1H),7.30-7.37(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.80(t,1H),5.83(s,2H),4.73-4.81(m,1H),4.51(br.s.,2H),4.29(br.s.,2H),2.57(s,3H),1.00-1.05(m,2H),0.82-0.88(m,2H)。

Compound 123

A solution of 2-aminocyclohexanecarboxylic acid (1.2 eq), triethylamine (10 eq) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq) in DMF was stirred at 110 ℃ for 1H. The crude product was purified by reverse phase HPLC to give the desired compound (3.4mg, 12% yield) as a white solid. ¹H NMR(500MHz,DMSO-d₆)δppm 8.22(d,1H),7.65(s,1H),7.30-7.37(m,1H),7.18-7.29(m,2H),7.11(t,1H),6.84(t,1H),5.82(s,2H),4.57(br.s.,1H),2.90(d,1H),2.58(s,3H),2.04(d,1H),1.81-1.92(m,1H),1.61-1.74(m,2H),1.36-1.55(m,4H)。

Compound 124

The target compound is prepared by 2 steps:

step 1: synthesis of 1- (3- (4- (3-aminoazetidin-1-yl) -5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone

A solution of tert-butyl azetidin-3-ylcarbamate (1.5 equiv.), triethylamine (10 equiv.) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equiv.) in DMF was stirred at 130 ℃ for 1 hour. The crude product was purified by reverse phase HPLC to give the desired intermediate 1- (3- (4- (3-aminoazetidin-1-yl) -5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (33mg, 99% yield) as a solid. The compound was taken to the next step without further purification.

Step 2: synthesis of Compound 124

To a stirred solution of ethyl isocyanate (1.5 equivalents) in toluene was added triethylamine (1 equivalent) and 1- (3- (4- (3-aminoazetidin-1-yl) -5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone. The mixture was stirred and heated at 90 ℃ for 48 hours, then concentrated in vacuo. The resulting crude residue was purified by reverse phase HPLC to give the desired compound (6.0mg, yield 15%) as a white solid. 1H NMR (500MHz, CDCl) ₃)δppm 7.91(br s,1H),7.30(br s,1H),7.10(br t,1H),6.91-7.04(m,3H),5.83(br d,2H),4.81(br s,1H),4.42-4.58(m,1H),4.32(br s,2H),3.14-3.22(m,2H),2.45-2.50(m,2H),2.45(s,3H),1.12-1.19(m,1H),1.08(br t,3H)。

Compound 125

A solution of 2-aminoethanol (10 equivalents), triethylamine (10 equivalents) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) in 1, 4-dioxane was stirred at 90 ℃ for 5 hours. The crude product was purified by reverse phase HPLC to give the desired compound (19.5mg, 91% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm8.23(d,1H),7.97(br.s.,1H),7.71(s,1H),7.30-7.36(m,1H),7.20-7.26(m,1H),7.11(t,1H),6.82(t,1H),5.83(s,2H),3.57-3.63(m,4H),2.58(s,3H)。

A solution of 5- (trifluoromethyl) -1,3, 4-thiadiazol-2-amine (1.5 equivalents), triethylamine (10 equivalents) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) in 1, 4-dioxane was stirred at 90 ℃ for 5 hours. The crude product was purified by reverse phase HPLC to give the desired compound (10.4mg, 38% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.76(br.s.,1H),7.80(s,1H),7.31-7.40(m,2H),7.16-7.23(m,2H),7.10-7.15(m,1H),5.89(s,2H),2.63(s,3H)。

Compound 193

A solution of 3-amino-2-hydroxy-2-methylpropanamide (1.5 equivalents), triethylamine (10 equivalents) and 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 equivalent) in 1, 4-dioxane was stirred at 90 ℃ for 5 hours. The crude product was purified by reverse phase HPLC to afford the desired compound.¹H NMR(500MHz,DMSO-d₆)δppm 8.24(d,1H),7.69(s,1H),7.38(br.s.,1H),7.30-7.35(m,2H),7.19-7.26(m,2H),7.11(t,1H),6.84(t,1H),5.83(s,2H),3.76(dd,1H),3.59(dd,1H),2.57(s,3H),1.29(s,3H)。

Compound 128

The target compound is prepared by 5 steps:

step 1: synthesis of N-methoxy-N-methylisoxazole-3-formamide

To a solution of isoxazole-3-carboxylic acid (2.0g, 1.0 eq) in dichloromethane (80ml) was added oxalyl chloride (2.0ml, 1.3 eq) with cooling at 0 ℃ followed by two drops of DMF. The mixture was stirred at room temperature for 1 hour. To this mixture was added N, O-dimethylhydroxylamine hydrochloride (2.2g, 1.3 equivalents) followed by triethylamine (8.6ml, 3.5 equivalents). The mixture was stirred at room temperature for 3 hours. The mixture was quenched with 1N HCl (50mL) and diluted with DCM (50 mL). The layers were separated and the aqueous layer was extracted with DCM (2X 50 mL). The organics were combined, washed with water (2X 50mL), brine (50mL), and MgSO ₄Drying and filtering. The solvent was removed in vacuo to give the crude product. Purification by silica gel chromatography eluting with a gradient of EtOAc/hexanes afforded N-methoxy-N-methylisoxazole-3-carboxamide as a yellow oil (3.21g, 93% yield).¹H NMR(400MHz,CDCl₃)δ8.46-8.51(m,1H),6.67-6.76(m,1H),3.80(br.s.,3H),3.39(br.s.,3H)。

Step 2: synthesis of ethyl 4- (isoxazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoate

To a cooled solution of N-methoxy-N-methylisoxazole-3-carboxamide (0.31g, 1.0 eq.) and ethyl propionate (0.39g, 2.0 eq.) in anhydrous THF (10mL) at-55 deg.C was added as a 1M solution of bis (trimethylsilyl) in THF (3.8mL, 1.9 eq.). The mixture was stirred at-40 ℃ for 20 minutes. A black solution was obtained. The mixture was quenched with 1N HCl (4mL) and warmed to room temperature. Mixing the mixtureIn EtOAc (10mL) and H₂Partition between O (6 mL). The organic layer was washed with 15% NaCl and concentrated in vacuo to give an oil. Purification by column chromatography eluting with a 0 to 100% EtOAc/hexanes gradient afforded 4- (isoxazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoic acid ethyl ester (769mg, 43% yield);¹H NMR(500MHz,CDCl₃)δ8.42(d,1H),6.77(d,1H),6.19(s,1H),4.47(q,2H),3.76(s,3H),3.22(s,3H),1.41(t,3H)。

step 3: synthesis of ethyl 5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazole-3-carboxylate

A solution of 4- (isoxazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoic acid ethyl ester (67mg, 1.0 eq) and (3,3,4,4, 4-pentafluorobutyl) hydrazine hydrochloride (56mg, 1.0 eq) in ethanol (1.3ml) was stirred at 65 ℃ for 1 h. The mixture was concentrated in vacuo. The residual oil was purified by column chromatography eluting with a gradient of 0 to 10% ethyl acetate in hexanes to give ethyl 5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazole-3-carboxylate (49mg, yield 53%) as a light yellow solid. ¹H NMR(500MHz,CDCl₃)δ8.54(d,1H),7.27(s,1H),6.62(d,1H),4.96-5.01(m,2H),4.42-4.48(m,2H),2.73-2.86(m,2H),1.41-1.45(m,3H)。

Step 4: synthesis of 5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazole-3-carboxamidine

In a 40ml vial, a suspension of ammonium chloride (218mg, 6.5 equivalents) in toluene (2.1ml) was cooled at 0 ℃ for 30 minutes under argon. To this mixture was added 2.0M trimethylaluminum in toluene (2.0ml, 6.5 equivalents). The mixture was removed from the ice bath and stirred at room temperature until foaming ceased. The mixture became clear. To this mixture was added 5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazole-3-carboxylic acid ethyl ester (223mg, 1.0 eq) in toluene (2.0 ml). The mixture was heated at 110 ℃ for 24 hours. Gases generated during the reaction are released. The mixture was cooled to 0 ℃, diluted with toluene and quenched with methanol. The mixture was stirred vigorously and the precipitate formed was removed by filtration using a buchner funnel. The filtrate was transferred to a round bottom flask and concentrated in vacuo to give a solid. The solid was then treated with 5:1 EtOAc: IPA mixture (60ml) and saturated sodium bicarbonate solution (40 ml). The aqueous layer was back-extracted with a 5:1 mixture of EtOAc and IPA (50 mL). The organic layers were combined, dried over magnesium sulfate, filtered and evaporated in vacuo. The solid was dried to give 5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazole-3-carboxamidine (230mg, quantitative yield) as a brown solid. ¹H NMR(400MHz,DMSO-d₆)δppm 9.18(d,1H)7.54(s,1H)7.08(d,1H)4.88(t,2H)2.76-3.04(m,2H)。

Step 5: synthesis of Compound 128

A solution of 5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazole-3-carboxamidine (106mg, 1.0 eq), DBU (87. mu.l, 1.8 eq) and ethyl 3- (dimethylamino) -2-fluoroacrylate (133mg, 2.5 eq) in EtOH (1.6mL) was heated at 70 ℃ for 24H. The mixture was concentrated in vacuo to give the crude product as an oil. Purification by column chromatography eluting with a 0 to 100% ethyl acetate/hexanes gradient gave 5-fluoro-2- (5- (isoxazol-3-yl) -1- (3,3,4,4, 4-pentafluorobutyl) -1H-pyrazol-3-yl) pyrimidin-4 (3H) -one (4.5mg, 4% yield) as a white solid.¹H NMR(400MHz,CD₃OD)δ8.86(d,1H),8.05(d,1H),7.43(s,1H),6.98(d,1H),5.00-5.09(m,2H),2.83-3.01(m,2H)。

Compound 129

Will contain triethylamine (3.0 equivalent)Amount), a solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 32mg, 1.0 eq) and 2-methylbutane-1, 2-diamine (3.0 eq) in NMP (0.5ml) was stirred at room temperature for 24 hours. The mixture was diluted with ethyl acetate (50ml) and washed with water (50 ml). The organic layer was dried, filtered and evaporated to give the crude product as an oil. The oil was purified by column chromatography eluting with a gradient of 0 to 10% MeOH in DCM to give 1- (3- (4- ((2-amino-2-methylbutyl) amino) -5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (12mg, 32% yield) as a white solid. ¹H NMR(500MHz,METHANOL-d₄)δppm 8.24(d,1H)7.77(s,1H)7.28-7.36(m,1H)7.25(d,1H)7.13-7.19(m,1H)7.08(t,1H)5.94(s,2H)3.72(s,2H)2.56-2.61(m,3H)1.72-1.83(m,2H)1.37(s,3H)1.08(t,3H)。

Compound 130

A solution of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) isoxazole (intermediate-1A, 100mg, 1.0 eq), triethylamine (8.0 eq) and 2- (((S) -3-amino-1, 1, 1-trifluoro-2-methylpropan-2-yl) amino) -2-phenylethanol hydrochloride (3.0 eq) in DMF (1.3ml) was stirred at room temperature for 24H. The mixture was diluted with ethyl acetate (50ml) and washed with water (50 ml). The organic layer was dried, filtered and evaporated to give the crude product as an oil. The oil was purified by column chromatography eluting with a 0 to 100% EtOAc/hexanes gradient to give 2-phenyl-2- (((S) -1,1, 1-trifluoro-3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-methylpropan-2-yl) amino) ethanol (132mg, 82% yield) as a white solid.¹H NMR(500MHz,DMSO-d₆)δppm 8.25(d,1H)7.76(br.s.,1H)7.45(s,1H)7.32(d,3H)7.17-7.24(m,4H)7.11-7.16(m,1H)7.09(t,1H)6.88(t,1H)5.88(s,2H)5.24(t,1H)3.85(dd,1H)3.70(dd,1H)3.21(td,1H)2.91(d,1H)1.04(s,3H)。

Compound 131

A mixture containing 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) isoxazole (intermediate-1A, 420mg, 1.0 eq), triethylamine (6.0 eq) and 2- (((R) -3-amino-1, 1, 1-trifluoro-2-methylpropan-2-yl) amino) -2-phenylethanol hydrochloride (3.0 eq) was stirred. ) Was stirred at room temperature for 24 hours. The mixture was diluted with ethyl acetate (50ml) and washed with water (50 ml). The organic layer was dried, filtered and evaporated to give the crude product as an oil. The oil was purified by column chromatography eluting with a 0 to 100% EtOAc/hexanes gradient to give 2-phenyl-2- (((R) -1,1, 1-trifluoro-3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -2-methylpropan-2-yl) amino) ethanol (348mg, 52% yield) as a white solid. ¹H NMR (500MHz, methanol-d)₄)δppm 8.76(s,1H)8.08(d,1H)7.32-7.46(m,3H)7.24(t,3H)7.18(d,2H)7.06-7.12(m,1H)7.02(s,1H)6.88(s,1H)5.96(s,2H)4.17(dd,1H)3.82-4.09(m,2H)3.48-3.56(m,1H)3.36(d,1H)1.08-1.18(m,3H)。

Compound 132

A solution of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) isoxazole (intermediate-1A, 364mg, 1.0 eq), (4R, 6R) -tert-butyl-6- (2-aminoethyl) -2, 2-dimethyl-1, 3-dioxane-4-acetate (3.0 eq) and triethylamine (3.0 eq) in 1, 4-dioxane (3.7ml) and water (1.3ml) was heated at 60 ℃ for 2 hours. The mixture was diluted with ethyl acetate (50ml) and washed with water (50 ml). The organic layer was dried, filtered and evaporated in vacuo to give the crude product as an oil. The oil was purified by column chromatography eluting with a gradient of 0 to 50% ethyl acetate in hexanes to give the desired compound as a white solid (536mg, 90% yield).¹H NMR (500MHz, chloroform-d) delta ppm 8.34(s,1H)7.97(s,1H)7.23(s,1H)7.01-7.08(m,1H)6.86-6.91(m,1H)6.83(t,1H)6.72(t,1H)6.46(s,1H)5.87(s,2H)4.00(qd,2H)3.73-3.82(m,1H)3.46-3.54(m,1H)2.29-2.36(m,1H)2.18-2.25(m,1H)1.64-1.81(m,2H)1.43-1.52(m,2H)1.30-1.37(m,15H)。

Compound 133

To a solution of compound 132(0.575g, 1.0 eq) in DCM (100mL) was added TFA (14mL, 200 eq). The mixture was stirred at room temperature for 1 hour. The mixture was concentrated in vacuo. The resulting residue was partitioned between DCM (50ml) and 1N sodium bicarbonate (50 ml). The organic layer was dried, filtered and evaporated to give an oil. The oil was purified by column chromatography eluting with a 0 to 100% ethyl acetate/hexanes gradient to give (4R, 6R) -6- (2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) ethyl) -4-hydroxytetrahydro-2H-pyran-2-one (365mg, 78% yield) as a white solid. ¹H NMR (500MHz, chloroform-d) delta ppm 8.45-8.48(m,1H)8.10(d,1H)7.36(s,1H)7.17-7.24(m,1H)7.01-7.07(m,1H)6.98(td,1H)6.88(td,1H)6.71(d,1H)5.92-6.03(m,2H)5.59(br.s.,1H)4.86-4.93(m,1H)3.68-3.97(m,2H)2.53-2.73(m,2H)2.32(dt,1H)2.06-2.11(m,1H)1.97-2.04(m,1H)1.72(ddd, 1H).

Compound 134

A solution of compound 133(173mg, 1.0 eq) and sodium hydroxide (1.0 eq) in THF (0.9ml) and MeOH (0.9ml) was stirred at room temperature for 1 hour. The mixture was concentrated in vacuo to give (3R, 5R) -7- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3, 5-dihydroxyheptanoic acid sodium salt (187mg, yield 100%) as a white solid.¹H NMR(500MHz,MeOD)δ8.72(d,1H),7.98(d,1H),7.40(s,1H),7.18-7.27(m,1H),7.01-7.08(m,1H),6.98(t,1H),6.88(d,1H),6.74(t,1H),5.91(s,2H),4.08-4.15(m,1H),3.93(dt,1H),3.64-3.86(m,2H),2.25-2.40(m,2H),1.82-1.97(m,2H),1.62-1.80(m,2H)。

Compound 135

A solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq), 4-aminobutyric acid (2 eq) and triethylamine (10 eq) in dry dioxane was heated at 90 ℃ for 1 day. The resulting mixture was concentrated and the crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to give 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) butyric acid (2.2mg, 9% yield) as a white solid. ¹H NMR (500MHz, methanol-d 4) Δ ppm 8.27(d,1H),7.96(s,1H),7.32(m,1H),7.10(s,2H),6.97(s,1H),5.99(s,2H),3.85(t,2H),2.65(m,3H),2.49(s,2H),2.07(m, 2H).

Compound 136

A solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 1 eq), 4, 4-dimethylpyrrolidine-3-carboxylic acid (2 eq) and triethylamine (10 eq) in dry dioxane was heated at 90 ℃ for 1 day. The resulting mixture was concentrated and the crude product was purified by reverse phase HPLC eluting with a 30-80% acetonitrile in 0.1% formic acid gradient to give 1- (2- (5-acetyl-1- (2-fluorobenzyl) -1H-pyrazol-3-yl) -5-fluoropyrimidin-4-yl) -4, 4-dimethylpyrrolidine-3-carboxylic acid (2.7mg, 10% yield) as a white solid.¹H-NMR (500MHz, methanol-d 4) Δ ppm8.20(m,1H),7.77(s,1H),7.31(m,1H),7.10(m,2H),6.88(m,1H),5.95(s,2H),4.22(m,2H),3.94(m,1H),3.72(m,1H),3.03(m,1H),2.61(s,3H),1.38(s,3H),1.17(s, 3H).

Compound 137

A solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (1 equivalent), (1R, 2R) -2-aminocyclohexanecarboxylic acid (2 equivalents) and triethylamine (10 equivalents) in dry dioxane was heated at 90 ℃ for 1 day. The resulting mixture was concentrated and the crude product was purified by reverse phase HPLC using a gradient of 30-80% acetonitrile in 0.1% formic acid to give (1R, 2R) -2- ((2- (5-acetyl-1- (2-fluorobenzyl) -1H-pyrazol-3-yl) -5-fluoropyrimidin-4-yl) amino) cyclohexanecarboxylic acid (2.8mg, 10% yield) as a white solid. ¹H-NMR (500MHz, methanol-d 4) delta ppm 8.00(m,1H),7.74(m,1H),7.29(m,1H),7.08(m,2H),6.80(m,1H),5.92(s,2H),4.54(m,1H),2.60(s,3H),2.49(m,1H),2.10(m,2H),1.84(m,2H),1.69(m,1H),1.56(m,1H),1.37(m, 2H).

Compound 138

A solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (1 equivalent), (S) -3-amino-4-methylpentanoic acid (2 equivalents) and triethylamine (10 equivalents) in dry dioxane was heated at 90 ℃ for 1 day. The resulting mixture was concentrated and the crude product was purified by reverse phase HPLC eluting with a 30-80% acetonitrile in 0.1% formic acid gradient to give (S) -3- ((2- (5-acetyl-1- (2-fluorobenzyl) -1H-pyrazol-3-yl) -5-fluoropyrimidin-4-yl) amino) -4-methylpentanoic acid (5.4mg, 20% yield) as a white solid.¹H-NMR (500MHz, methanol-d 4) delta ppm 8.00(m,1H),7.72(m,1H),7.28(m,1H),7.09(m,2H),6.81(m,1H),5.92(s,2H),4.73(m,1H),2.59(s,5H),2.07(m,1H),1.04(t, 6H).

Compound 139

A solution of 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (1 eq), 4-phenylpiperidine-4-carboxylic acid (3 eq) and triethylamine (10 eq) in dry dioxane was heated at 90 ℃ for 1 day. The resulting mixture was concentrated and the crude product was purified by reverse phase HPLC Purification using a gradient of 30-80% acetonitrile in 0.1% formic acid afforded 1- (2- (5-acetyl-1- (2-fluorobenzyl) -1H-pyrazol-3-yl) -5-fluoropyrimidin-4-yl) -4-phenylpiperidine-4-carboxylic acid (7.9mg, 27% yield) as a white solid.¹H-NMR (500MHz, methanol-d 4) Δ ppm 8.31(m,1H),7.92(m,1H),7.51(m,2H),7.39(m,2H),7.32(m,2H),7.11(m,2H),6.95(m,1H),5.98(m,2H),4.83(m,2H),3.70(m,2H),2.81(m,2H),2.64(s,3H),2.16(m, 2H).

Compound 185

The target compound is prepared by 5 steps:

step 1: cyanopyrimidines

A solution of zinc (II) cyanide (10.8g, 92mmol) and 2-chloro-5-fluoro-4-methoxypyrimidine (15.0g, 92mmol) in dimethylformamide (200mL) was bubbled with nitrogen at room temperature for 10 minutes. Tetrakis (triphenylphosphine) palladium (0) (10.0g, 8.65mmol) was added, degassing was continued for 10 min and the reaction was heated at 90 ℃ for 2 days. The mixture was cooled to room temperature and diluted with ethyl acetate (150mL), brine (50mL) and concentrated aqueous ammonium hydroxide (10 mL). After mixing, the layers were separated and the aqueous phase was extracted with another portion of ethyl acetate (150 mL). The combined organic phases were washed with 2 × 20mL brine, then dried over sodium sulfate, filtered and concentrated by rotary evaporation at 60 ℃. In SiO₂Purification as above using a gradient elution with hexanes/ethyl acetate afforded the chloropyrimidine starting material recovered and 5.5g of the desired cyanopyrimidine as a colorless oil. The recovered starting material was reprocessed as described above to give 3.0g of the intermediate (total yield 8.5g, yield 60%).

¹H-NMR(500MHz,CDCl3)δ8.41(s,1H),4.17(s,3H)ppm。

Step 2: pyrimidine esters

Will be described in detail1 (8.1g, 52.9mmol) was cooled in ice and 1N sodium hydroxide (aq) (63.5mL, 63.5mmol) was added over 5 minutes. The mixture was stirred at room temperature overnight, then 3N hydrochloric acid (aq) was added to pH 3 while being cooled again in ice. The mixture was concentrated to dryness, first by rotary evaporation, then high vacuum, to give 12.8g of crude carboxylic acid as a white solid, which was directly esterified. The crude solid was stirred in dry methanol (150mL) at room temperature and concentrated sulfuric acid (1.5mL, 29.1mmol) was added. The mixture was stirred overnight, then cooled in ice, then 10% NaHCO was added₃Aqueous solution (100mL) was then stirred at room temperature for an additional 1 hour. The solvent was removed in vacuo and the residue partitioned between water (100mL) and ethyl acetate (300 mL). 3X 20mL of H was used for the organic phase₂O washes, then the combined aqueous phases are back-extracted with 200mL ethyl acetate. The combined organic phases are washed with Na₂SO₄Dried, filtered and concentrated by rotary evaporation. In SiO₂Purification as above using hexanes/ethyl acetate as a gradient eluent gave the ester pyrimidine intermediate as a white solid (4.5g, 46% yield from cyanopyrimidine).

¹H-NMR(500MHz,CDCl₃)δ8.45(s,1H),4.20(s,3H),4.03(s,3H)ppm。

Step 3 and step 4: intermediate-17

Step 3: a solution of 1- (isoxazol-3-yl) ethanone (4.0g, 36.3mmol) in THF (200mL) was cooled in dry ice/acetone. Lithium bis (trimethylsilyl) amide (1M in toluene, 33.8mL, 33.8mmol) was added over 10 minutes, followed by stirring at-65 ℃ to-70 ℃ for 30 minutes. A solution of the above-obtained pyrimidine ester (4.5g, 24.2mmol) in THF (20mL) was added dropwise to the solution of valeric acid over 5 minutes and stirring was continued at room temperature overnight. The solvent was removed in vacuo, and the residue was dissolved in ether (100mL) and filtered. The filter cake was washed with diethyl ether (20mL) and air dried to give 8.2g of crude diketoisoxazole, which was taken directlyThe next reaction was carried out without further purification. LCMS (M/e)266(M + H).

Step 4: crude diketoisoxazole (theoretical 6.4g, 24.2mmol) was dissolved in methanol (100mL), glacial acetic acid (11.4mL, 199mmol) and hydrazine hydrate (4.0mL, 83mmol) were added and the solution was heated at 60 ℃ for 30 min. The solvent was removed under vacuum, and the residue was taken up in ethyl acetate (50mL) and 10% NaHCO₃The aqueous solution (200mL) was dissolved and stirred at room temperature until no more gas evolution was observed. Hexane (80mL) was added and the biphasic mixture was stirred for 30 min and filtered. The filter cake was washed with 2X 50mL of H ₂O, 1:1 hexane/ethyl acetate (50mL) and dried under vacuum to give 2.69g of intermediate-17 as a light brown solid. The organic filtrate was found to contain impure product. The mixture was washed with SiO using dichloromethane/ethyl acetate as gradient eluent₂Purification by chromatography gave 0.39g of intermediate-17 (total yield: 3.1g, 71% from the ester pyrimidine).¹H-NMR(500MHz,CD₃OD)δ8.76(s,1H),8.49(s,1H),7.40(s,1H),6.94(s,1H),4.23(s,3H)ppm.LCMS(m/e)262(M+H)。

Step 5: compound 185

A solution of tert-butyl 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (intermediate-17, 1 eq) and lithium tert-butoxide (2 eq) in dimethoxyethane (2ml) was stirred at 60 ℃ for 5 min. 1- (bromomethyl) -4-toluene (1.1 eq) was added and the reaction stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed under nitrogen. The resulting solid was dissolved in methanol (0.5ml) and concentrated aqueous HCl (140ul) and stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to afford compound 185(1.5mg, 5% yield) as a white solid.¹H NMR (500MHz, methanol-d 4) delta ppm 8.75(m,1H),7.95(m,1H),7.35(s,1H),7.11(m,4H),6.85(m,1H),5.84(s,2H),2.28 (s,3H)。

Compound 187

A solution of tert-butyl 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (intermediate-17, 1 eq) and lithium tert-butoxide (3 eq) in dimethoxyethane (2ml) was stirred at 60 ℃ for 5 min. 2- (bromomethyl) pyridine, HBr (1.1 equiv.) were added thereto, and the reaction was stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed under nitrogen. The resulting solid was dissolved in methanol (0.5ml) and concentrated aqueous HCl (140ul) and stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to give compound 187(1.5mg, 5% yield) as a white solid.¹H NMR (500MHz, methanol-d 4) Δ ppm 8.78(s,1H),8.70(d,1H),8.19(t,1H),8.07(d,1H),7.69(t,1H),7.56(s,1H),7.52(d,1H),6.92(s,1H),6.20(s, 2H).

Compound 189

A solution of tert-butyl 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (intermediate-17, 1 eq) and lithium tert-butoxide (3 eq) in dimethoxyethane (2ml) was stirred at 60 ℃ for 5 min. 3- (bromomethyl) pyridine, HBr (1.1 equiv.) were added thereto, and the reaction was stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed under nitrogen. The resulting solid was dissolved in methanol (0.5ml) and concentrated aqueous HCl (140ul) and stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to afford compound 189(12.9mg, 40% yield) as a white solid. ¹H NMR (500MHz, methanol-d 4) delta ppm 8.90(s,1H),8.86(d,1H),8.77(d,1H),8.50(d,1H),8.08(d,1H),7.96(br.s.,1H),7.52(s,1H),7.00(d,1H), 6.14(s),2H)。

Compound 190

A solution of tert-butyl 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (intermediate-17, 1 eq) and lithium tert-butoxide (2 eq) in dimethoxyethane (2ml) was stirred at 60 ℃ for 5 min. 5- (bromomethyl) -3-methylisoxazole (1.1 eq.) was added and the reaction stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed under nitrogen. The resulting solid was dissolved in methanol (0.5ml) and concentrated aqueous HCl (140ul) and stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to give compound 190(13.2mg, 42% yield) as a white solid.¹H NMR (500MHz, methanol-d 4) Δ ppm 8.84(d,1H),8.04(m,1H),7.47(s,1H),6.97(d,1H),6.15(m,1H),6.06(s,2H),2.22(m, 3H).

Synthesis of Compound 141 and Compound 140

A solution of 2- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) oxazole, 2- (aminomethyl) -1,1,1,3,3, 3-hexafluoropropan-2-ol (intermediate-1F, 3.0 equiv.) and triethylamine (10 equiv.) in dioxane-water (2:1) was heated at 90-100 ℃ for 5 days. The reaction mixture was diluted with water, acidified to pH 4 with 1N HCl solution and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed in vacuo. Purification by silica gel chromatography (gradient elution with 10-25% ethyl acetate in hexanes) afforded compound 141 as a white solid (38mg, 5% yield, 55% in 2 steps), and compound 140 as a white solid (11mg, 21%, 2 steps).

Compound 141:¹H-NMR(500MHz,CDCl₃)δppm 8.27(m,2H),7.73(s,1H),7.45(s,1H),7.23(m,1H),7.14(app.t,1H),7.05-7.00(m,2H),6.10(s,2H),5.72(br s,1H),4.15(d,2H). No exchangeable OH protons were observed.

Compound 140:¹H-NMR(500MHz,CDCl₃)δppm 8.13(d,1H),7.68(s,1H),7.48(s,1H),7.21(s,1H),7.18(m,1H),7.01(app.t,1H),6.95(app.t,1H),6.87(app.t,1H),6.09(s,2H),3.68(q,4H),1.29(t,6H)。

synthesis of Compound 142

To a THF gray-yellow suspension of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2, 3-difluorobenzyl) -1H-pyrazol-5-yl) isoxazole (intermediate-1G, using the corresponding 2- (1- (2, 3-difluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) which had been described in patent application publication WO2013/101830 as starting material, analogously to the preparation of intermediate 1F) and zinc powder (2.5 equivalents), acetic acid (2.8 equivalents) was added and the reaction mixture was heated at 75 ℃ for 2 days, after cooling to room temperature, poured into a 1N NaOH solution, extracted with ethyl acetate, the organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (gradient elution 10-50% ethyl acetate in hexanes) to give compound 142(39mg, 74%) as a white solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.12(s,1H),8.97(s,2H),7.69(s,1H),7.38(m,1H),7.30(s,1H),7.14(m,1H),6.78(app.t,1H),5.97(s,2H)。

Compound 143

The target compound is synthesized by 5 steps:

step 1: 1- ((3-Fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylic acid ethyl ester and

synthesis of ethyl 1- ((3-fluoropyridin-2-yl) methyl) -3- (isoxazol-3-yl) -1H-pyrazole-5-carboxylate

To a suspension of 3-fluoro-2- (hydrazinomethyl) pyridine hydrochloride (1.0 eq) and potassium carbonate (0.5 eq) in ethanol/water (10:1) was added ethyl 4- (isoxazol-3-yl) -2- (methoxy (methyl) amino) -4-oxobut-2-enoate (1 eq). The resulting orange suspension was heated at 60 ℃ for 24 hours. The crude mixture was concentrated in vacuo. Water was added and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed in vacuo. Purification by silica gel chromatography (gradient elution of 10-25% ethyl acetate in hexane) afforded ethyl 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylate (47%) and ethyl 1- ((3-fluoropyridin-2-yl) methyl) -3- (isoxazol-3-yl) -1H-pyrazole-5-carboxylate (9.7%).

Step 2: synthesis of 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylic acid

To a solution of ethyl 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylate in THF/water (3:1 ratio) was added lithium hydroxide (2.0 equiv.). After 5 hours, the reaction mixture was concentrated in vacuo to remove most of the THF. The resulting mixture was diluted with water and acidified to pH 4-5 by addition of 1N HCl solution. The product, 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylic acid, was collected by vacuum filtration. The filtrate was extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and the solvent removed in vacuo to yield additional product (96%).

Step 3: synthesis of 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carbonitrile

To a suspension of 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxylic acid, 2-methylpropan-2-amine (3.0 eq) and triethylamine (2.0 eq) in ethyl acetate was added n-propylphosphonic anhydride (T3P, 50% by weight in ethyl acetate, 3.0 eq). The resulting yellow solution was heated at 65 ℃ for 3 hours. The solvent was removed in vacuo. Phosphorus trichloride (20 equivalents) was added and the resulting mixture was stirred at 70 ℃ for 2 hours. The reaction was quenched by careful pouring into a mixture of water and ice, neutralized to pH 7 by addition of saturated sodium bicarbonate solution/solid sodium bicarbonate, and extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed in vacuo to yield 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carbonitrile (> 99%).

Step 4: synthesis of 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine (intermediate-18)

To a solution of 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carbonitrile in methanol was added sodium methoxide (0.5N in MeOH, 4.0 eq) and stirred for 4H. Ammonium chloride (10 equivalents) was added. The reaction mixture was stirred at ambient temperature for 36 hours and at 50 ℃ for 6.5 hours. The reaction was concentrated in vacuo and partitioned between half-saturated sodium bicarbonate solution and ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo to give 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine (98%), which was used without further manipulation.

Step 5: synthesis of Compound 143

To a solution of 1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine (intermediate-18) in ethanol was added sodium (Z) -3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (4.0 eq) and heated at 90 ℃ for 2H. After cooling to room temperature, the reaction mixture was neutralized by adding HCl (1.25M in EtOH). The resulting suspension was concentrated in vacuo. The residue was partitioned between dichloromethane/isopropanol (7:1) and water and the pH was adjusted to 6 by addition of 1N NaOH solution. The aqueous layer was back-extracted with dichloromethane/isopropanol (7: 1). The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed in vacuo. Purification by silica gel chromatography (gradient elution of 5-20% acetonitrile/methanol (7:1) in dichloromethane) afforded compound 143(220mg, 60%) as a light brown solid.¹H-NMR(500MHz,DMSO-d₆)δppm 13.2(br s,1H),9.07(s,1H),8.23(d,1H),8.12(br s,1H),7.76(app.t,1H),7.63(s,1H),7.40(m,1H),7.23(s,1H),6.05(s,2H)。

Compound 144

The target compound is synthesized by 2 steps:

step 1: synthesis of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- ((3-fluoropyridin-2-yl) methyl) -1H-pyrazol-5-yl) isoxazole

A solution of 5-fluoro-2- (1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4 (3H) -one in phosphorus trichloride (85 eq.) as solvent was heated at 65 ℃ for 2 hours. The reaction mixture was cooled to ambient temperature, blown dry under nitrogen and then concentrated twice from toluene. The resulting yellowish brown solid, 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- ((3-fluoropyridin-2-yl) methyl) -1H-pyrazol-5-yl) isoxazole, was dried in vacuo and used in the next step without further work-up.

Step 2: synthesis of Compound 144

3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- ((3-fluoropyridin-2-yl) methyl) -1H-pyrazol-5-yl) isoxazole and zinc powder (1.7 eq) were made up in THF as a pale yellow suspension, acetic acid (2.8 eq) was added and heated at 75 ℃ for 3H. An additional amount of zinc dust (2.8 equiv.) and acetic acid (2.8 equiv.) were added and the reaction was heated at 75 ℃ for 20 hours. After cooling to room temperature, the reaction mixture was filtered and the filtrate was partitioned between half-saturated sodium bicarbonate solution and ethyl acetate. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (30-60% ethyl acetate/hexanes gradient elution) to give compound 144(30mg, 35%, 2 steps) as a pale yellow solid.

¹H-NMR(500MHz,DMSO-d₆)δppm 9.06(s,1H),8.95(s,2H),8.25(d,1H),7.76(app.t,1H),7.64(s,1H),7.40(m,1H),7.28(s,1H),6.06(s,2H)。

Compound 145

Adding 1- ((3-Fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine (intermediate-18) to a pyridine solution of 1, 8-diazabicyclo [5.4.0]Undec-7-ene (DBU, 4.0 equiv.) and 3-ethoxypropionitrile(2.5 equivalents) and heated at 110 ℃ for 46 hours. The reaction mixture was cooled to ambient temperature, concentrated in vacuo, and partitioned between half-saturated sodium bicarbonate solution and dichloromethane. The organic phase was dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (10-25% acetonitrile/methanol (7:1) in dichloromethane gradient) followed by preparative HPLC (5-75% acetonitrile/water gradient with 0.1% trifluoroacetic acid) to give compound I-145(17mg, 21%, TFA salt) as a white solid. ¹H-NMR(500MHz,DMSO-d₆)δppm14.1(br s,1H,TFA),9.11(s,1H),8.78(br s,2H),8.24(d,1H),8.10(d,1H),7.78(app.t,1H),7.61(s,1H),7.42(m,1H),7.28(s,1H),6.63(d,1H),6.11(s,2H)。

Compound 146

The target compound is synthesized by 2 steps:

step 1: synthesis of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- ((3-fluoropyridin-2-yl) methyl) -1H-pyrazol-5-yl) isoxazole

A solution of 5-fluoro-2- (1- ((3-fluoropyridin-2-yl) methyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4 (3H) -one in phosphoryl trichloride (100 eq) was heated at 65 ℃ for 3 hours. The reaction mixture was cooled to ambient temperature, blown dry under nitrogen and then concentrated twice from toluene. The resulting yellowish brown solid, 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- ((3-fluoropyridin-2-yl) methyl) -1H-pyrazol-5-yl) isoxazole, was dried in vacuo and used in the next step without further work-up.

Step 2: synthesis of Compound 146

A solution of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- ((3-fluoropyridin-2-yl) methyl) -1H-pyrazol-5-yl) isoxazole and ammonium hydroxide (35 eq, 29% in water) in dioxane was heated at 60 ℃ for 22H. The resulting yellow suspension was diluted with water. The product was collected by filtration, washed with water and dried in vacuo to give compound 146(37mg, 98% over 2 steps) as a light brown solid.¹H-NMR(500MHz,DMSO-d₆)δppm 9.04(s,1H),8.25(d,1H),8.21(d,1H),7.75(app.t,1H),7.44(s,1H),7.42-7.34(m,3H),7.22(s,1H),6.00(s,2H)。

Compound 147

A suspension of intermediate-1A (50.0mg, 0.134mmol), 4-methoxy-1H-pyrrol-2 (5H) -one (18.2mg, 0.161mmol) and cesium carbonate (65.4mg, 0.201mmol) in dioxane (2mL) was heated at 95 ℃ for 2H and then at 70 ℃ for 12H. The reaction mixture was then diluted in water, extracted with dichloromethane (3 × 30mL), dried (sodium sulfate), filtered and concentrated to give a residue. Purification by reverse phase HPLC using a gradient of 5 to 95% acetonitrile/water (containing 0.1% trifluoroacetic acid) over 25 min afforded 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) -4-methoxy-1H-pyrrol-2 (5H) -one as a white solid, compound 147(2.0mg, 3% yield). ¹H NMR(500MHz,CDCl₃)δ(ppm):8.64(d,1H),8.49(d,1H),7.36(s,1H),7.20–7.23(m,1H),7.02–7.07(m,1H),6.98–7.01(m,1H),6.87–6.90(m,1H),6.61(d,1H),5.99(s,2H),5.26(s,1H),4.70(s,2H),3.94(s,3H)。

Compound 148

A solution of intermediate-1A (70.0mg, 0.187mmol), (R) -2-amino-3-methylbutan-1-ol (0.0250mL, 0.225mmol) and triethylamine (0.104mL, 0.749mmol) in dioxane (1mL) and water (0.5mL) was stirred at 85 ℃ for 16 h. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate, which was filtered and dried. The crude product was dissolved in dichloromethane and washed with water (2X 30mL), dried (sodium sulfate), filtered and concentrated to give (R) -2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -3-methylbutan-1-ol, which was reacted to giveCompound 148(69.4mg, 95% yield) as a white solid. No purification is required.¹H NMR(500MHz,CD₃OD)δ(ppm):8.75(s,1H),8.05(d,1H),7.40(s,1H),7.24-7.29(m,1H),7.07-7.11(m,1H),7.01-7.04(m,1H),6.90(s,1H),6.79-6.82(m,1H),5.96(s,2H),4.34-4.37(m,1H),3.80(dd,1H),3.72(dd,1H),2.03-2.09(m,1H),1.05(d,3H),1.00(d,3H)。

Compound 149

A solution of intermediate-1A (70.0mg, 0.187mmol), (R) -2-amino-4-methylpentan-1-ol (0.0290mL, 0.225mmol) and trimethylamine (0.104mL, 0.749mmol) in dioxane (1mL) and water (0.5mL) was stirred at 85 ℃ for 16 h. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate, which was filtered and dried. The crude product was dissolved in dichloromethane and washed with water (2X 30mL), dried (sodium sulfate), filtered and concentrated to give (R) -2- ((5-fluoro-2- (1- (2-5-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -4-methylpent-1-ol, compound 149(67.3mg, 79% yield) as a white solid. No purification is required. ¹H NMR(500MHz,DMSO-d₆) δ (ppm) 9.09(s,1H),8.14(d,1H),7.43(s,1H),7.28-7.34(m,2H),7.19-7.23(m,2H,2 overlay shift), 7.08-7.11(m,1H),6.86-6.89(m,1H),5.91(d,1H),5.84(d,1H),4.74(m,1H),4.44(br.s,1H),3.46-3.50(m,1H),3.40-3.45(m,1H),1.56-1.62(m,1H),1.45-1.51(m,1H),1.36-1.41(m,1H),0.90(d,3H),0.88(d, 3H).

Compound 150

A solution of intermediate-1A (70.0mg, 0.187mmol), (R) - (+) -methyl mercaptan (30.4mg, 0.225mmol) and triethylamine (0.104mL, 0.749mmol) in dioxane (1mL) and water (0.5mL) was reacted at 85 ℃ for 16 h. The reaction mixture was then cooled to room temperature and diluted in water to form a yellow precipitate, which was filtered and dried. Will be coarseThe product was dissolved in dichloromethane and washed with water (2X 30mL), dried (sodium sulfate), filtered and concentrated to give (R) -2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) -4- (methylthio) butan-1-ol, compound 150(47.5mg, yield 54%) as a light yellow waxy solid. No purification is required.¹H NMR(500MHz,DMSO-d₆) δ(ppm):9.09(d,1H),8.17(d,1H),7.49(s,1H),7.39(d,1H),7.31-7.34(m,1H),7.23(s,1H),7.19-7.23(m,1H),7.08-7.11(m,1H),6.81-6.84(m,1H),5.90(d,1H),5.87(d,1H),4.80-4.82(m,1H),4.35-4.42(m,1H),3.51-3.55(m,1H),3.45-3.50(m,1H),2.50-2.54(m,2H),2.02(s,3H),1.90-1.95(m,1H),1.80-1.85(m,1H)。

Compound 151

A solution of intermediate-1A (75.0mg, 0.201mmol), (R) -2-aminohex-1-ol (28.2mg, 0.241mmol) and triethylamine (0.104mL, 0.749mmol) in dioxane (1mL) and water (0.5mL) was reacted at 85 ℃ for 16 hours. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate, which was filtered and dried. The crude product was dissolved in dichloromethane and washed with water (2 × 30mL), dried (sodium sulfate), filtered and concentrated to give (R) -2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) hex-1-ol, compound 151(83.2mg, 91% yield) as a white solid. No further purification was required.

¹H NMR(500MHz,DMSO-d₆) δ (ppm) 9.09(d,1H),8.15(d,1H),7.46(s,1H),7.29-7.34(m,2H),7.20-7.23(m,2H,2 overlay shift), 7.08-7.11(m,1H),6.81-6.85(m,1H),5.91(d,1H),5.87(d,1H),4.71-4.74(m,1H),4.29-4.34(m,1H),3.48-3.52(m,1H),3.42-3.48(m,1H),1.61-1.68(m,1H),1.46-1.53(m,1H),1.19-1.36(m,4H),0.81(t, 3H).

Compound 152

A solution of intermediate-1A (75.0mg, 0.201mmol), (R) -2-aminopentan-1-ol (24.8mg, 0.241mmol) and triethylamine (0.104mL, 0.749mmol) in 1mL dioxane and water (0.5mL) was stirred at 85 ℃ for 16 hours. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate, which was filtered and dried. The crude product was dissolved in dichloromethane and washed with water (2 × 30mL), dried (sodium sulfate), filtered and concentrated to give (R) -2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) pentan-1-ol, compound 152(47.1mg, yield 53%) as a white solid. No further purification was required.

¹H NMR(500MHz,DMSO-d₆) δ (ppm) 9.09(s,1H),8.15(d,1H),7.46(s,1H),7.29-7.35(m,2H),7.20-7.23(m,2H,2 overlay shift), 7.08-7.11(m,1H),6.83-6.86(m,1H),5.91(d,1H),5.87(d,1H),4.73(m,1H),4.32-4.38(m,1H),3.48-3.52(m,1H),3.43-3.47(m,1H),1.58-1.65(m,1H),1.46-1.53(m,1H),1.26-1.40(m,2H),0.88(t, 3H).

Compound 127

A solution of intermediate-1A (82.0mg, 0.219mmol), 2-amino-6, 6, 6-trifluorohexanol-1-ol (45.0mg, 0.263mmol) and triethylamine (0.122mL, 0.876mmol) in dioxane (1mL) and water (0.5mL) was heated at 85 ℃ for 72 hours. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate, which was filtered and dried. The crude product was dissolved in dichloromethane and washed with water (2 × 30mL), dried (sodium sulfate), filtered and concentrated to give 6,6, 6-trifluoro-2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) hex-1-ol, compound 127(80.1mg, 72% yield) as a white solid. No further purification was required.¹H NMR(500MHz,DMSO-d₆) Delta (ppm) 9.08(s,1H),8.18(d,1H),7.49(s,1H),7.38(d,1H),7.30-7.34(m,1H),7.20-7.24(m,2H,2 overlay shift), 7.07-7.10(m,1H),6.78-6.81(m,1H),5.88(s,2H),4.80(m,1H),4.33-4.38(m,1H),3.50-3.55(m,1H),3.45-3.49(m,1H),2.40-2.48(m,1H),2.20-2.29(m,1H),1.70-1.76(m,1H),1.54-1.63(m,2H),1.46-1.52(m,1H)。

Compound 153

A solution of intermediate-1A (74.5mg, 0.199mmol), 2-ethylbutane-1, 2-diamine (27.8mg, 0.239mmol) and triethylamine (0.111mL, 0.797mmol) in dioxane (1mL) and water (0.5mL) was heated at 85 ℃ for 16 h. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate which was filtered and dried in vacuo to give 2-ethyl-N1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) butane-1, 2-diamine as compound 153(76.8mg, 85% yield) as an off-white solid. No further purification was required. ¹H NMR(500MHz,CD₃OD)δ(ppm):8.77(s,1H),8.15-8.19(m,1H),7.46-7.49(m,1H),7.26-7.30(m,1H),7.10-7.13(m,1H),7.02-7.06(m,1H),6.90(s,1H),6.76-6.80(m,1H),5.98(s,2H),3.66(s,2H),1.59-1.72(m,4H),0.96-1.03(m,6H)。

Compound 154

A solution of intermediate-1A (70.0mg, 0.187mmol), 2, 3-dimethylbutane-1, 2-diamine (26.1mg, 0.225mmol) and triethylamine (0.104mL, 0.749mmol) in dioxane (1mL) and water (0.5mL) was heated at 85 ℃ for 16 h. The reaction mixture was then cooled to room temperature and diluted in water to form a white precipitate which was filtered and dried in vacuo to give N1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) -2, 3-dimethylbutane-1, 2-diamine as compound 154(70.7mg, 83% yield) as an off-white solid. No further purification was required.¹H NMR(500MHz,DMSO-d₆)δ(ppm):9.09(m,1H),8.18(d,1H),7.45(s,1H),7.31-7.35(m,1H),7.25-7.29(m,1H),7.19-7.23(m,2H),7.09-7.12(m,1H),6.88-6.91(m,1H),5.87(s,2H),3.41-3.49(m,2H)1.57-1.63(m,1H),0.91(d,3H),0.90(s,3H),0.88(s,3H), [2N-H protons were not observed]。

Compound 155

A solution of intermediate-1A (113mg, 0.303mmol), 2-methylpentane-1, 2-diamine (42.3mg, 0.364mmol) and triethylamine (0.169mL, 1.21mmol) in dioxane (1mL) and water (0.5mL) was heated at 85 ℃ for 16 hours. The reaction mixture was cooled to room temperature, diluted with water, extracted with dichloromethane (3 × 30mL), dried (sodium sulfate), filtered and concentrated to a viscous oil, which solidified upon standing to give N1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) -2-methylpentane-1, 2-diamine (117mg, 85% yield) as a white foamy solid. No further purification was required. ¹H NMR(500MHz,CD₃OD) δ(ppm):8.76(s,1H),8.07(d,1H),7.43(s,1H),7.24-7.29(m,1H),7.07-7.11(m,1H),7.01-7.04(m,1H),6.90(s,1H),6.80-6.83(m,1H),5.96(s,2H),3.67(d,1H),3.60(d,1H),1.42-1.50(m,4H),1.15(s,3H)。

Compound 156

A solution of intermediate-1A (101mg, 0.271mmol), 2, 4-dimethylpentane-1, 2-diamine (42.4mg, 0.326mmol) and triethylamine (0.151mL, 1.21mmol) in dioxane (1mL) and water (0.5mL) was heated at 85 ℃ for 16 hours. The reaction mixture was cooled, diluted in water, extracted with dichloromethane (3 × 30mL), dried (sodium sulfate), filtered and concentrated in vacuo to give N1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) -2, 4-dimethylpentane-1, 2-diamine as an off-white viscous gum as compound 156(109.7mg, 86% yield). No further purification was required.¹H NMR(500MHz,DMSO-d₆) Delta (ppm) 9.09(s,1H),8.18(d,1H),7.47(s,1H),7.31-7.34(m,1H),7.19-7.23(m,2H,2 overlay shift), 7.08-7.11(m,1H),6.85-6.88(m,1H),588(s,2H),3.36-3.44(m,2H),1.78-1.85(m,1H),1.58(br.s,2H),1.23-1.31(m,2H),1.01(s,3H),0.92(d,3H),0.88(d,3H), [2N-H protons were not observed]。

Compound 157

To a solution of (2R, 3S) -1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) -3-methylpiperidine-2-carboxylic acid (which compound was previously described in patent application publication WO2014144100, 25.0mg, 0.0520mmol) in dioxane (1mL) was added 4- (2-bromoethyl) morpholine hydrobromide (15.7mg, 0.0570mmol) followed by cesium carbonate (25.4mg, 0.0780 mmol). The reaction mixture was heated at 90 ℃ for 2H, then diluted with DMSO (1mL) and water (0.5mL) and directly purified by reverse phase HPLC using a gradient of 5 to 95% aqueous acetonitrile spiked with 0.1% trifluoroacetic acid over 25 min to give ethyl (2R, 3S) -2-morpholinoethyl 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) -3-methylpiperidine-2-carboxylate, compound 157(16.7mg, 54% yield) as a white foamy solid. ¹H NMR(500MHz,CD₃CN) delta (ppm) of 8.69(s,1H),8.24(d,1H),7.47(s,1H),7.30-7.34(m,1H),7.11-7.15(m,1H),7.08-7.11(m,1H),6.96-6.99(m,1H),6.90(s,1H),5.88(s,2H),5.32(d,1H),4.39-4.47(m,3H,2 overlay shift), 3.62-3.79(m,4H),3.50-3.55(m,2H),3.28-3.34(m,4H),2.84-2.93(m,2H),1.85-1.88(m,1H),1.68-1.75(m,2H),1.50-1.56(m,1H), 1.16H (d, 3H).

Compound 158

To a solution of 2-morpholinoethyl carbamate (134mg, 0.769mmol) in methanol (4mL) was added potassium tert-butoxide (86.0mg, 0.769 mmol). The reaction mixture was stirred at room temperature for 1 hour, then concentrated to give a white solid. The resulting solid was dissolved in DMSO (4mL) before the intermediate was added-1A (287mg, 0.769 mmol). The reaction mixture was stirred at room temperature for 72 hours, and then the dioxane was removed in vacuo. The crude product mixture was purified by reverse phase HPLC eluting with a 5 to 95% acetonitrile/water (spiked with 0.1% trifluoroacetic acid) gradient over 25 minutes to give a mixture of the two compounds. Further purification by silica gel chromatography eluting with a gradient of 3 to 15% methanol in dichloromethane over 50 min afforded 2-morpholinoethyl (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) carbamate, compound 158(28.7mg, 7% yield) as a white crystalline solid. ¹H NMR(500MHz,CD₃OD) delta (ppm) 8.78(s,1H),8.64(d,1H),7.53(s,1H),7.26-7.30(m,1H),7.08-7.12(m,1H),7.02-7.06(m,1H),6.89(s,1H),6.85-6.89(m,1H),5.97(s,2H),4.51-4.56(m,2H),3.78-3.86(m,4H),3.33-3.37(m,2H), [4H ] are not observed, and CD is not observed₃OD synchronization]。

Compound 159

A solution of intermediate-1A (281mg, 0.751mmol), 2-amino-5- (aminomethyl) phenol dihydrochloride (182mg, 0.864mmol) and triethylamine (0.524mL, 3.76mmol) in dioxane (3mL) and water (1.5mL) was heated at 90 ℃ for 16 hours. The reaction mixture was then cooled to room temperature, diluted in 1N aqueous hydrochloric acid and water, filtered and dried in vacuo to give a crude solid. Purification was performed using silica gel chromatography eluting with a gradient of 3 to 10% methanol in dichloromethane over 60 minutes to give a mixture of products. Further purification by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 45 min afforded 2-amino-5- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) phenol, compound 159(135mg, 38% yield) as an orange solid.¹H NMR(500MHz,DMSO-d₆)δ(ppm):9.09(s,1H),8.93(br.s,1H),8.17(d,1H),8.11(m,1H),7.49(s,1H),7.31-7.35(m,1H),7.24(s,1H),7.20-7.24(m,1H),7.10-7.13(m,1H),6.85-6.88(m,1H),6.67(s,1H),6.62(d,1H),6.50(d,1H),5.90(s,2H),4.51(d,2H),4.42(br.s,2H)。

Compound 160

To a solution of compound 159(49.5mg, 0.104mmol) in pyridine (1mL) was added cyclopropanesulfonyl chloride (0.0120mL, 0.115 mmol). After 16H, the reaction mixture was directly purified by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 min to give N- (4- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxyphenyl) cyclopropanesulfonamide compound 160(41.9mg, 69% yield) as an orange solid. ¹H NMR(500MHz,CD₃CN)δ(ppm):8.65(s,1H),8.10(d,1H),7.49(br.s,1H),7.36(s,1H),7.27-7.30(m,1H),7.27(d,1H),7.10-7.13(m,1H),7.05-7.09(m,1H),7.02(m,2H),6.91-6.94(m,2H),6.84(s,1H),6.60-6.62(m,1H),5.86(s,2H),4.70(d,2H),2.43-2.48(m,1H),0.89-0.94(m,2H),0.85-0.89(m,2H)。

Compound 161

To a solution of compound 159(40.8mg, 0.086mmol) in pyridine (1mL) was added methanesulfonyl chloride (7.36. mu.L, 0.0940 mmol). After 16H, the reaction mixture was concentrated to about 10% of its volume and purified by reverse phase HPLC eluting with a gradient of 5 to 95% acetonitrile/water spiked with 0.1% trifluoroacetic acid over 25 min to give N- (4- (((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) methyl) -2-hydroxyphenyl) methanesulfonamide as compound 161(5.8mg, yield 12%) as a brown solid.¹H NMR(500MHz,CD₃CN) delta (ppm) of 8.69(s,1H),8.10(d,1H),7.61(m,1H),7.49(s,1H),7.26-7.30(m,1H),7.26(d,1H),7.16(br.s,1H),7.03-7.11(m,3H,3 chemical shift overlap), 6.94-6.97(m,2H),6.88(s,1H),5.86(s,2H),4.77(d,2H),2.89(s,3H), [1N-H protons are not observed]。

Compound 162

A suspension of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionic acid (intermediate-22, 50.0mg, 0.117mmol, which is described in patent application publication WO 2014144100), 4- (2-bromoethyl) morpholine hydrobromide (35.5mg, 0.129mmol) and cesium carbonate (57.3mg, 0.176mmol) in dioxane (1mL) was heated at 90 ℃ for 16 hours. The reaction mixture was cooled to room temperature and then purified by silica gel chromatography with gradient elution over 45 min using 3 to 7% methanol in dichloromethane to give 2-morpholino) ethyl 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propanoate as a white solid, compound 162(34.8mg, 55% yield. ¹H NMR(500MHz,CD₃OD)δ(ppm):8.76(s,1H),8.07(d,1H),7.44(s,1H),7.25-7.29(m,1H),7.07-7.11(m,1H),7.02-7.05(m,1H),6.91(s,1H),6.81-6.84(m,1H),5.96(s,2H),4.22(t,2H),3.91(t,2H),3.63(t,4H),2.76(t,2H),2.59(t,2H),2.45-2.50(m,4H)。

Compound 163

To a mixed solution of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionic acid (intermediate-22, 52.1mg, 0.122mmol) in diethyl ether (4mL) and methanol (1mL) was added a 2M solution of trimethylsilyldiazomethane (183. mu.L, 0.367mmol) in diethyl ether. After 30 min, the reaction mixture was concentrated and then purified by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 45 min to give methyl 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionate as compound 163(27.1mg, 50% yield) as a white solid.¹H NMR(500MHz,CD₃OD)δ(ppm):8.76(s,1H),8.06(d,1H),7.43(s,1H),7.25-7.29(m,1H),7.07-7.11(m,1H),7.02-7.05(m,1H),6.90(s,1H),6.80-6.83(m,1H),5.96(s,2H),3.89(t,2H),3.66(s,3H),2.74(t,2H)。

Compound 164

To a mixed solution of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionic acid (intermediate-22, 68.6mg, 0.161mmol) in dichloromethane (4mL) and acetonitrile (2mL) was added 2M oxalyl chloride (0.201mL, 0.402mmol) at 0 ℃. Three drops of N, N-dimethylformamide were added and the resulting reaction mixture was stirred at 0 ℃ for 10 minutes and then warmed to room temperature. After 30 min, additional oxalyl chloride solution (0.8mL) was added, then the reaction mixture was stirred for 15 min, then concentrated to dryness to give 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionyl chloride (72mg, 0.162mmol, 101% yield) as a waxy off-white solid.

To a solution of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionyl chloride (36mg, 0.081mmol) in dichloromethane (7.5mL) was added propan-2-ol (0.616mL, 8.09 mmol). The reaction mixture was stirred for 15 minutes, then the solvent was removed in vacuo to give the crude product. Purification was performed by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 45 min to give isopropyl 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionate as compound 164(11mg, yield 29%) as an off-white waxy solid.¹H NMR(500MHz,CDCl₃)δ(ppm):8.46(s,1H),8.15(s,1H),7.35(s,1H),7.18-7.22(m,1H),7.01-7.05(m,1H),6.96-6.99(m,1H),6.83-6.86(m,1H),6.60(s,1H),5.98(s,2H),5.74(br.s,1H),5.08(m,1H),3.93-3.96(m,2H),2.70(t,2H),1.27(d,6H)。

Compound 165

To a mixed solution of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionic acid (intermediate-22, 68.6mg, 0.161mmol) in dichloromethane (4mL) and acetonitrile (2mL) was added 2M oxalyl chloride (0.201mL, 0.402mmol) at 0 ℃. Three drops of N, N-dimethylformamide were added and the resulting reaction mixture was stirred at 0 ℃ for 10 minutes and then warmed to room temperature. After 30 min, additional oxalyl chloride solution (0.8mL) was added, then the reaction mixture was stirred for 15 min, then concentrated to dryness to give 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionyl chloride (72mg, 0.162mmol, 101% yield) as a waxy off-white solid.

To a solution of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionyl chloride (36mg, 0.081mmol) in dichloromethane (7.5mL) was added anhydrous ethanol (0.473mL, 8.09 mmol). The reaction mixture was stirred for 15 minutes, then the solvent was removed in vacuo to give the crude product. Purification by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 45 min afforded ethyl 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) propionate as compound 165(8.0mg, 22% yield) as an off-white waxy solid.¹H NMR(500MHz,CDCl₃) Delta (ppm) 8.46(s,1H),8.16(s,1H),7.39(br.s,1H),7.18-7.22(m,1H),7.01-7.05(m,1H),6.96-6.99(m,1H),6.84-6.87(m,1H),6.61(s,1H),5.98(s,2H),4.20(q,2H),3.95-3.98(m,2H),2.74(t,2H),1.29(t,3H), and [1NH protons are not observed]。

Compound 166

A mixed solution of intermediate-1A (294mg, 0.787mmol) and N-methylprop-2-en-1-amine (0.187mL, 1.98mmol) in dioxane (2mL) and water (1mL) was heated at 90 ℃ for 2 hours. The reaction mixture was cooled to room temperature and then diluted with 1N hydrochloric acid solution and water, and a precipitate formed. Filtering the solid and vacuum drying to obtain To N-allyl-5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -N-methylpyrimidin-4-amine, compound 166(288mg, yield 90%) as a light yellow solid.¹H NMR(500MHz,CDCl₃)δ(ppm):8.45(d,1H),8.16(d,1H),7.29(s,1H),7.17-7.21(m,1H),7.00-7.05(m,1H),6.95-6.98(m,1H),6.83-6.87(m,1H),6.59(d,1H),5.97(s,2H),5.87-5.96(m,1H),5.25-5.28(m,1H),5.23(s,1H),4.23(d,2H),3.28(d,3H)。

Compound 167

A mixed solution of 3- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) isoxazole (intermediate-1A, 195mg, 0.521mmol) and trans-pyrrolidine-3, 4-diol (113mg, 1.10mmol) in dioxane (1mL) and water (0.5mL) was heated at 90 ℃ for 2 hours. The reaction mixture was then cooled to room temperature and diluted with 1N hydrochloric acid and water to form a white precipitate, which was filtered and dried. The crude product was dissolved in dichloromethane/isopropanol (5:1) and washed with water (2X 30mL), saturated sodium chloride solution (2X 30mL), dried (sodium sulfate), filtered and concentrated to give trans-1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) pyrrolidine-3, 4-diol, compound 167(226mg, 98% yield) as an off-white solid.¹H NMR(500MHz,DMSO-d₆)δ(ppm):9.08(d,1H),8.22(d,1H),7.52(s,1H),7.31-7.35(m,1H),7.24(d,1H),7.20-7.23(m,1H),7.09-7.12(m,1H),6.83-6.86(m,1H),5.90(s,2H),5.21(d,2H),4.05(br.s,2H),3.79-3.83(br.m,2H),3.68(d,2H)。

Compound 168

A mixed solution of intermediate-1A (122mg, 0.326mmol), 4- (1-hydroxy-2-methylamino-ethyl) -benzene-1, 2-diol (62.1mg, 0.339mmol) and triethylamine (0.182mL, 1.31mmol) in dioxane (1mL) and water (0.5mL) was heated at 90 ℃ for 2 hours. Then mixing the reaction The mixture was cooled to room temperature, diluted in 1N aqueous hydrochloric acid and water, filtered and dried to give the crude product as a tan solid. This material was purified by silica gel chromatography, eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 minutes, to give 4- (2- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) (methyl) amino) -1-hydroxyethyl) benzene-1, 2-diol, compound 168(24.4mg, yield 14%) as a brown solid.¹H NMR(500MHz,CD₃OD)δ(ppm):8.75(d,1H),8.07(d,1H),7.38(s,1H),7.24-7.28(m,1H),7.06-7.10(m,1H),7.01-7.04(m,1H),6.90(d,1H),6.87-6.89(m,1H),6.84-6.86(m,1H),6.73-6.75(m,1H),6.72(d,1H),5.96(s,2H),4.89-4.93(m,1H),3.82-3.93(m,2H),3.30(d,3H)。

Compound 169

A mixed solution of intermediate-1A (115mg, 0.308mmol), cis-pyrrolidine-3, 4-diol (41.2mg, 0.400mmol) and triethylamine (0.214mL, 1.538mmol) in dioxane (1mL) and water (0.5mL) was heated at 90 ℃ for 2 hours. The reaction mixture was then cooled to room temperature, diluted with 1N aqueous hydrochloric acid and water, filtered and dried in vacuo to give the crude cis-1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) pyrrolidine-3, 4-diol, compound 169(104.6mg, 0.219mmol, 71.0% yield) as an off-white solid. No further purification was required.¹H NMR(500MHz,DMSO-d₆)δ(ppm):9.08(d,1H),8.21(d,1H),7.51(s,1H),7.30-7.35(m,1H),7.24(d,1H),7.20-7.23(m,1H),7.09-7.12(m,1H),6.82-6.86(m,1H),5.90(s,2H),5.01(d,2H),4.12-4.15(m,2H),3.80-3.84(m,2H),3.55-3.59(m,2H)。

Compound 170

intermediate-1A (65.5mg, 0.175mmol) and 2- (piperazin-1-yl) ethanol (0.0860mL, 0.701mmol) were combined A mixed solution of dioxane (2mL) and water (1mL) was heated at 85 ℃ for 12 hours. The reaction mixture was then cooled to room temperature, diluted with 1N aqueous hydrochloric acid and water, filtered and dried in vacuo to give 2- (4- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) piperazin-1-yl) ethanol, compound 170(40.8mg, yield 50%) as a white solid. No purification is required.¹H NMR(500MHz,CD₃OD)δ(ppm):8.74(d,1H),8.15(d,1H),7.42(s,1H),7.24-7.28(m,1H),7.06-7.10(m,1H),7.00-7.03(m,1H),6.89(d,1H),6.79-6.82(m,1H),5.94(s,2H),3.96(t,4H),3.72(t,2H),2.67(t,4H),2.59(t,2H)。

Compound 171

A mixed solution of intermediate-1A (70.0mg, 0.187mmol) and methyl 4-piperidinecarboxylate (0.0760mL, 0.562mmol) in dioxane (2mL) and water (1mL) was heated at 90 ℃ for 12 hours. The reaction mixture was cooled to room temperature, then diluted with water, extracted with dichloromethane (3 × 30mL), dried (sodium sulfate), filtered and concentrated to give the crude product. Purification by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 min afforded ethyl 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl)) -1H-pyrazol-3-yl) pyrimidin-4-yl) piperidine-4-carboxylate, compound 171(73.8mg, 82% yield) as a clear viscous oil which solidified to a white gum upon standing.¹H NMR(500MHz,CD₃OD)δ(ppm):8.75(d,1H),8.14(d,1H),7.42(s,1H),7.24-7.29(m,1H),7.08-7.10(m,1H),7.01-7.04(m,1H),6.90(d,1H),6.80-6.83(m,1H),5.95(s,2H),4.58-4.61(m,2H),3.70(s,3H),3.27-3.31(m,2H),2.72-2.78(m,1H),2.03-2.06(m,2H)1.74-1.82(m,2H)。

Compound 172

To 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole- 3-yl) pyrimidin-4-yl) (methyl) amino) propionic acid (intermediate-23, 25.6mg, 0.0580mmol, which is described in patent application publication WO 2014144100) in diethyl ether (0.75mL) and methanol (0.25mL) was added a 2M solution of trimethylsilyldiazomethane (0.035mL, 0.070mmol) in diethyl ether. After 1H, the reaction was purified by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 min to give methyl 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) (methyl) amino) propionate as a clear oil 172(7.2mg, 27% yield) which solidified to a white waxy solid upon standing.¹H NMR(500MHz,CDCl₃)δ(ppm):8.45(d,1H),8.18(d,1H),7.30(s,1H),7.18-7.21(m,1H),7.01-7.04(m,1H),6.95-6.99(m,1H),6.85-6.89(m,1H),6.59(d,1H),5.96(s,2H),4.00(t,2H),3.69(s,3H),3.35(d,3H),2.76(t,2H)。

Compound 173

To a solution of 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) piperidin-4-ol (intermediate-24, 15mg, 0.034mmol, which is described in patent application publication WO 2014144100) in dichloromethane (1mL) was added N-. alpha. -t-Boc-glycine (7.8mg, 0.044mmol), followed by N, N-dimethylaminopyridine (2.1mg, 0.017mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (8.5mg, 0.044 mmol). The reaction mixture was stirred at room temperature for 72 hours, then purified by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 minutes to give 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) piperidin-4-yl 2- ((tert-butoxycarbonyl) amino) acetate (21.0mg, 103% yield) as a white solid.

To a solution of 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) piperidin-4-yl 2- ((tert-butoxycarbonyl) amino) acetate (21.0mg, 0.035mmol) in dichloromethane (4mL) was added trifluoroacetic acid (0.30mL, 3.9 mmol). The reaction mixture was heated at 60 ℃ for 1 hour, then the reaction mixture was cooled to room temperature, neutralized by addition of saturated sodium bicarbonate solution, extracted with dichloromethane (3 × 30mL), dried (sodium sulfate), filtered and concentrated to give 1- (5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) piperidin-4-yl 2-aminoacetate as compound 173(12.7mg, 73% yield) as a pink solid.

¹H NMR(500MHz,CD₃OD)δ(ppm):8.75(d,1H),8.16(d,1H),7.42(s,1H),7.25-7.29(m,1H),7.07-7.11(m,1H),7.01-7.04(m,1H),6.90(d,1H),6.80-6.83(m,1H),5.95(s,2H),5.12-5.16(m,1H),4.18-4.24(m,2H),3.76-3.82(m,2H),3.43(s,2H),2.05-2.10(m,2H)1.77-1.84(m,2H)。

Compound 174

To a mixed solution of 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) butyric acid (intermediate-24, 8.6mg, 0.020mmol, which is described in patent application publication WO 2014144100) in diethyl ether (0.50mL) and methanol (0.167mL) was added a 2M solution of trimethylsilyldiazomethane (9.8. mu.L, 0.020mmol) in diethyl ether. After 1H, the reaction was concentrated and then purified using reverse phase HPLC eluting with a gradient of 5 to 95% acetonitrile/water spiked with 0.1% trifluoroacetic acid over 25 min to give methyl 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) butanoate, compound 174(2.8mg, 32% yield) as a clear colorless oil.

¹H NMR(500MHz,CDCl₃) Delta (ppm):8.52(d,1H),8.38(d,1H),7.48(s,1H),7.23-7.27(m,1H),7.14-7.18(m,1H),7.04-7.07(m,1H),7.01-7.04(m,1H),6.66(d,1H),5.93(s,2H),3.81(m,2H),3.72(s,3H),2.54(t,2H),2.07-2.12(m,2H), [ 1N-H) are not observed]。

Compound 175

To a mixed solution of 5- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) pentanoic acid (37.2mg, 0.0820mmol, which intermediate was previously described in patent application publication WO 2014144100) in diethyl ether (0.75mL) and methanol (0.250mL) was added a 2M solution of trimethylsilyldiazomethane in diethyl ether (0.045mL, 0.090 mmol). After 1H, the reaction was directly purified by silica gel chromatography, eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 min to give methyl 5- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) pentanoate, compound 175(12.8mg, yield 33%) as a clear colorless waxy gum.¹H NMR(500MHz,CDCl₃)δ(ppm):8.45(d,1H),8.14(d,1H),7.34(s,1H),7.17-7.22(m,1H),7.01-7.04(m,1H),6.95-6.98(m,1H),6.84-6.87(m,1H),6.65(d,1H),5.98(s,2H),5.17(m,1H),3.68(s,3H),3.62-3.67(m,2H),2.43(t,2H),1.74-1.81(m,4H)。

Compound 176

A mixed solution of intermediate-1A (124mg, 0.332mmol) and 4-aminobutan-1-ol (0.122mL, 1.33mmol) in dioxane (2mL) and water (1mL) was heated at 70 ℃ for 2 hours. The reaction mixture was diluted with water to form a white precipitate. The product was filtered and dried in vacuo to give 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) butan-1-ol as a white solid, compound 176(98.8mg, yield 70%). No purification is required.

¹H NMR(500MHz,CD₃OD)δ(ppm):8.74(s,1H),8.01(d,1H),7.40(s,1H),7.24-7.28(m,1H),7.06-7.10(m,1H),7.01-7.04(m,1H),6.89(m,1H),6.81-6.84(m,1H),5.95(s,2H),3.61-3.66(m,4H),1.74-1.80(m,2H)1.62-1.68(m,2H)。

Compound 177

To a solution of 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) -pyrimidin-4-yl) amino) propan-1-ol (intermediate-27, 98.5mg, 0.239mmol, which is described in patent application publication WO 2014144100), N-. alpha. -t-Boc-glycine (50.2mg, 0.287mmol) and N, N-dimethylaminopyridine (8.8mg, 0.072mmol) in dichloromethane (2mL) was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (54.9mg, 0.287 mmol). The reaction was stirred at room temperature for 16H, then the reaction mixture was directly purified by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 min to give butyl 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) 2- ((tert-butoxycarbonyl) amino) acetate intermediate (98.8 mg).

To a solution of butyl 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) 2- ((tert-butoxycarbonyl) amino) acetate in dichloromethane (2mL) was added trifluoroacetic acid (0.50mL, 6.5 mmol). The reaction mixture was heated at 60 ℃ for 30 minutes and then deprotected to completion. The reaction mixture was then neutralized by the addition of saturated sodium carbonate solution, extracted with dichloromethane (3X 30mL), dried (sodium sulfate), filtered and concentrated in vacuo to give propyl 3- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) 2-aminoacetate, compound 177(68.6mg, 61% yield) as a waxy white solid. No purification is required. ¹H NMR(500MHz,CD₃OD):δ(ppm)8.75(s,1H),8.04(d,1H),7.41(s,1H),7.24-7.29(m,1H),7.07-7.11(m,1H),7.02-7.05(m,1H),6.90(m,1H),6.82-6.85(m,1H),5.95(s,2H),4.28(t,2H),3.72(t,2H),3.36(s,2H),2.02-2.09(m,2H)。

Compound 178

To a solution of 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) butan-1-ol (compound 176, 69.1mg, 0.162mmol), N-. alpha. -t-Boc-glycine (34.1mg, 0.194mmol) and N, N-dimethylaminopyridine (5.9mg, 0.049mmol) in dichloromethane (2mL) was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (37.3mg, 0.194 mmol). The reaction was stirred at room temperature for 16H, then the reaction mixture was directly purified by silica gel chromatography eluting with a gradient of 1 to 8% methanol in dichloromethane over 60 min to give butyl 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) 2- ((tert-butoxycarbonyl) amino) acetate (64.9 mg).

To a solution of butyl 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) 2- ((tert-butoxycarbonyl) amino) acetate (64.9mg) in dichloromethane (2mL) was added trifluoroacetic acid (0.50mL, 6.5 mmol). The reaction mixture was heated at 60 ℃ for 30 minutes and then deprotected to completion. The reaction mixture was cooled to room temperature, neutralized by the addition of saturated sodium carbonate solution, extracted with dichloromethane (3 × 30mL), dried (sodium sulfate), filtered and concentrated to give butyl 4- ((5-fluoro-2- (1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) pyrimidin-4-yl) amino) 2-aminoacetate, compound 178(35.4mg, yield 45%) as a clear gum. No further purification was required.

Compound 179

Compound 179 is obtained by reacting 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 0.02, 0.057mmol), 4-isopropylpiperidine-4-carboxylic acid (0.054g, 0.315mmol) and triethylamine (10 equivalents) as a base in dioxane (0.5 mL). The reaction was completed after 15 hours at 100 ℃. After water treatment, the product was purified using RP-HPLC to give the product as a tan solid (0.012g, 39%).¹H NMR(500MHz,DMSO-d6)δppm 12.62(br.s.,1H),8.29(d,1H),7.33(q,1H)7.75(s,1H),7.18-7.27(m,1H),7.11(t,1H),6.81(t,1H),5.82(s,2H),4.53(d,2H),3.02(t,2H),2.58(s,3H),2.12(d,2H),1.72(dt,1H),1.41-1.55(m,2H),0.87(d,6H)。

Compound 180

By reacting 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 0.02, 0.057mmol), 3-aminopropane-1, 2-diol (0.052g, 0.57mmol) and triethylamine (10 equivalents) as a base in dioxane, the objective product (0.5mL) was obtained. The reaction was completed after 15 hours at 100 ℃. After water treatment, the product was purified using RP-HPLC, followed by flash chromatography eluting with 0-30% 7:1ACN: MeOH in DCM to afford the product as a white solid (0.014g, 54%).¹H NMR (500MHz, methanol-d 4) Δ ppm 8.08(d,1H),7.27(q,1H)7.68(s,1H),7.07-7.14(m,1H),7.04(t,1H),6.79(t,1H),5.90(s,2H),2.56(s,3H)3.87(quintet,1H),3.75-3.81(m,1H),3.61-3.68(m,1H),3.58(d,2H), -NH and-OH protons are exchanged with the solvent.

Compound 181

The desired product was obtained by reacting 1- (3- (4-chloro-5-fluoropyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazol-5-yl) ethanone (intermediate-1B, 0.02, 0.057mmol), 4-methylpiperidine-4-carboxylic acid (0.045g, 0.315mmol) and triethylamine (10 equivalents) as a base in dioxane (0.5 mL). The reaction was completed after 15 hours at 100 ℃. After water treatment, the product was purified using RP-HPLC to give the product as a tan solid (0.025g, 86%).¹H NMR (500MHz, methanol-d 4) delta ppm;¹H NMR(500MHz,DMSO-d6)δppm 12.34-12.73(br.s,1H),8.30(d,1H),7.74(s,1H),7.28-7.39(m,1H),7.17-7.27(m,1H),7.06-7.15(m,1H),6.81(t,1H),5.82(s,2H),4.20(d,2H),3.36(t,2H),2.58(s,3H),2.07(d,2H),1.50(t,2H),1.19(s,3H)。

intermediate-30

The intermediate is prepared by two steps:

step 1: commercial 3-fluoro-2-methylphenol (2.0g, 15.9mmol) and tert-butylchlorodimethylsilane (3.6g, 23.8mmol) were reacted in dichloromethane (20mL) at room temperature. Triethylamine (5.5mL, 39.6mmol) and 4-dimethylaminomethylpyridine (0.1g, 0.8mmol) were added and stirring continued at room temperature overnight. The mixture was diluted with ethyl acetate (200mL), washed with water (3 × 30mL) and brine, then dried over sodium sulfate, filtered and concentrated by rotary evaporation to give the product intermediate-29 as a colorless oil (3.85g, 101% yield).¹H-NMR(500MHz,CDCl₃)δ6.77(q,1H),6.43(t,1H),6.36(d,1H),1.90(d,3H),0.80(s,9H),0.00(s,6H)ppm。

Step 2: intermediate-29 (3.6g, 15.0mmol) and N-bromosuccinimide (2.8g, 15.7mmol) were combined in carbon tetrachloride (20mL) at room temperature. AIBN (0.2g, 1.5mmol) was added and the solution was heated at 80 ℃ for 2 hours. The mixture was cooled to room temperature and filtered. CCl for filter cake ₄Washed and the combined filtrates concentrated by rotary evaporation. The residue was passed through SiO₂Purification by chromatography eluting with a hexane/ethyl acetate gradient gave intermediate-30 (4.9g, 102% yield) as a colorless oil.¹H-NMR(500MHz,CDCl3)δ6.96(q,1H),6.50(t,1H),6.46(d,1H),4.38(s,2H),0.88(s,9H),0.12(s,6H)ppm。

Intermediate-31 and intermediate-32

The above compound was prepared from general intermediate-17:

3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (intermediate-17, 50mg, 0.19mmol) was dissolved in dimethoxyethane (3mL) at room temperature. A solution of potassium tert-butoxide in tert-butanol (1.0M, 0.38mL, 0.38mmol) and intermediate-30 (92)mg, 0.29mmol), the mixture is reacted at 60 ℃ for 1 hour. The cooled solution was diluted with ethyl acetate (80mL), washed with water (2 × 10mL), brine, then dried over sodium sulfate, filtered and concentrated by rotary evaporation. Using hexane/ethyl acetate as eluent in SiO₂And performing chromatographic separation to obtain two thick paste products. Intermediate-31 (21mg, 22% yield).¹H-NMR(500MHz,CDCl₃) Δ 8.38(d,1H),8.35(d,1H),7.53(s,1H),7.14(q,1H),6.6-6.7(m,3H),6.09(s,2H),4.13(s,3H),0.93(s,9H),0.23(s,6H) ppm intermediate-32 (24mg, 25% yield).¹H-NMR(500MHz,CDCl₃)δ8.47(d,1H),8.35(d,1H),7.28(s,1H),7.10(q,1H),6.55-6.65(m,3H),5.91(s,2H),4.14(s,3H),0.95(s,9H),0.26(s,6H)ppm。

Compound 182

To a solution of intermediate-32 (21mg, 0.04mmol) in methanol (1mL) was added concentrated aqueous hydrochloric acid (0.3mL, 3.0mmol) and the mixture was heated in a sealed vial at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo to give the product as a white solid (15mg, 96% yield). LCMS (M/e)372(M + H).

Compound 183

The compound is prepared by two steps:

step 1: a mixture of compound 182(15mg, 0.04mmol) and phosphorus oxychloride (0.50mL, 5.4mmol) was reacted at 60 ℃ for 2 hours, and then the solvent was removed under vacuum. By SiO₂Chromatography, eluting with a hexane/ethyl acetate gradient, afforded pure intermediate-33 (2mg, yield 16%) as a white solid.¹H-NMR(500MHz,CDCl₃)δ10.31(br s,1H),8.66(s,1H),8.60(d,1H),7.32(s,1H),7.19(q,1H),6.85(d,1H),6.70(d,1H),6.62(t,1H),5.97(s,2H)ppm。

Step 2: intermediate-33 (2mg, 0.005mmol and 2- (aminomethyl) -1,1,1,3,3, 3-hexafluoropropan-2-ol (10mg, 0.05mmol) were dissolved in dimethyl sulfoxide (1mL), heated overnight at 125 ℃ in a sealed vial, the reaction mixture diluted with ethyl acetate and washed with water (3 × 5mL), brine, dried over sodium sulfate, filtered and rotary evaporated to give a residue which was eluted with hexane/ethyl acetate over SiO₂Purification by chromatography gave compound 183 as a white solid (2mg, 64% yield).¹H-NMR(500MHz,CDCl₃)δ9.59(br s,1H),8.27(s,1H),8.24(br s,1H),7.18(s,1H),7.17(q,1H),6.83(s,1H),6.81(s,1H),6.68(s,1H),6.61(t,1H),5.93(s,2H),5.58(br t,1H),4.18(d,2H)ppm。

Intermediate-34

Intermediate-34 (colorless oil) was prepared in 44% overall yield using 3-fluoro-4-methylphenol as the starting material using the same conditions used for the synthesis of intermediate-30.¹H-NMR(500MHz,CDCl3)δ7.25(t,1H),6.63(dd,1H),6.58(dd,1H),4.53(s,2H),1.00(s,9H),0.24(s,6H)ppm。

Intermediate-36

The intermediate is prepared from an intermediate-17 through two steps:

step 1: 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (90mg, 0.34mmol) was dissolved in dimethoxyethane (3mL) at room temperature. A solution of potassium tert-butoxide in tert-butanol (1.0M, 0.69mL, 0.69mmol) and intermediate-35 (165mg, 0.52mmol) were added successively and the mixture was heated at 60 ℃ for 1 hour. The cooled solution was diluted with ethyl acetate (80mL), washed with water (2 × 10mL), brine, then dried over sodium sulfate, filtered and concentrated by rotary evaporation. SiO using hexane/ethyl acetate as eluent ₂Purifying by chromatography to obtain colorless glassProduct of (4) (38mg, 22% yield).¹H-NMR(500MHz,CDCl₃)δ8.47(s,1H),8.40(d,1H),7.33(s,1H),6.80(t,1H),6.52(dd,1H),6.46(dd,1H),5.88(s,2H),4.18(s,3H),0.94(s,9H),0.15(s,6H)ppm。

Step 2: to a solution of intermediate-35 (60mg, 0.12mmol) in methanol (1mL) was added concentrated aqueous hydrochloric acid (0.3mL, 3.0mmol) and the mixture was heated in a sealed vial at 60 ℃ overnight. The solvent was removed under vacuum and the demethylated residue (44mg, 99% yield) was directly subjected to the next reaction. LCMS (M/e)370 (M-H). The residue (44mg, 0.12mmol) was heated in phosphorus oxychloride (1.0mL, 10.7mmol) at 50 ℃ for 5 h, then the solvent was removed under vacuum. The residue was triturated with ether/hexanes and re-dried to give crude intermediate-36 as a white solid (14mg, 20% yield). Proceed to the next reaction without characterization.

Compound 184

Intermediate-36 (14mg, 0.036mmol) and 2- (aminomethyl) -1,1,1,3,3, 3-hexafluoropropan-2-ol (28mg, 0.14mmol) were dissolved in dimethyl sulfoxide (1mL), the solution was heated at 125 ℃ overnight, the reaction mixture was diluted with ethyl acetate (50mL), washed with water (3 × 5mL) and brine, dried over sodium sulfate, filtered and rotary evaporated to give a residue which was purified by preparative reverse phase HPLC using a gradient of water/acetonitrile (0.1% trifluoroacetic acid) as eluent to give the product as a white solid (2mg, 8% yield). ¹H-NMR (500MHz, d 6-acetone). delta.9.00 (br s,1H),8.93(s,1H),8.33(d,1H),7.80(br t,1H),7.40(s,1H),7.21(t,1H),7.05(s,1H),6.59(dd,1H),6.55(dd,1H),5.83(s,2H),4.18(d,2H) ppm.

Compound 207

The target compound was prepared in two steps:

step 1: synthesis of N' - (cyanomethyl) -1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carbohydrazide (intermediate-28)

To a suspension of 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine hydrochloride (1 equivalent, which is described in the previous patent application publication WO 2013/101830) in DMF was added hydrazine hydrate (1.5 equivalent), and the reaction was stirred at 23 ℃ for 1 hour. 2-bromoacetonitrile (4 equivalents) and triethylamine (5 equivalents) were then added and the reaction stirred at 23 ℃ for 2 hours. The solution was diluted with a mixture of 1:1 ethyl acetate and water. The layers were separated and the aqueous layer was extracted with ethyl acetate (2 times). The organic layers were combined and washed with water (3 times) and brine. The organic phase was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The resulting residue was purified by silica gel chromatography (using DCM/methanol solution) to give the desired intermediate, N' - (cyanomethyl) -1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carbohydrazide (397mg, 75% yield) as a yellow foam. ¹H NMR(400MHz,CDCl₃)δppm8.41-8.47(m,1H),7.17-7.28(m,1H),6.92-7.11(m,3H),6.75-6.87(m,1H),6.50-6.63(m,1H),5.86(s,2H),5.02(s,2H),3.90-4.06(m,3H)。

Step 2: synthesis of Compound 207

A solution of N' - (cyanomethyl) -1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazol-3-yl) carbohydrazide (1 eq, intermediate-28) in ethanol was heated at 70 ℃ for 40 min. The reaction mixture was poured into a 1:1 mixture of ethyl acetate and saturated ammonium chloride. The layers were separated and the aqueous layer was extracted 2 times with ethyl acetate. The organics were combined, dried over magnesium sulfate, filtered and the solvent removed in vacuo. The resulting residue was purified by reverse phase HPLC to give the desired compound (9mg, yield 7%) as a white solid.¹H NMR(400MHz,CD₃OD)δppm 8.82(d,1H),8.31(s,1H),7.62(s,1H),7.23-7.35(m,1H),6.88-7.16(m,4H),5.96-6.08(m,2H)。

Compound 198

Sodium ethoxide (7.8 equivalents) and compound 207(1 equivalent) were suspended in methanol and microwaved at 150 ℃ for 30 minutes. The solvent was removed in vacuo and the crude residue diluted with ethyl acetate and saturated aqueous ammonium chloride. The layers were separated and the aqueous layer was extracted 2 times with ethyl acetate, dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by reverse phase HPLC (5-75% acetonitrile/water/0.1% trifluoroacetic acid, gradient elution for 20 min) afforded the desired compound, compound 198(6mg, 23% yield) as a brown solid.¹H-NMR(500MHz,MeOD)δppm 8.36(s,1H),7.89(s,1H),7.36-7.32(m,1H),7.16-7.09(m,2H),7.00-6.97(s,1H),6.01(s,2H),2.61(s,3H)。

Compound 199

Intermediate-20 (1 equivalent, the synthesis of this compound is described in patent application publication WO2014/144100), 4-dimethylaminopyridine (0.1 equivalent), and 2- ((tert-butoxycarbonyl) amino) acetic acid (1.5 equivalent) were suspended in dichloromethane at 0 ℃ and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.9 equivalent) was added. After 10 minutes, the solution was warmed to 23 ℃ and then stirred for 15 hours. The reaction mixture was diluted with dichloromethane and washed with 1N aqueous hydrochloric acid. The organics were dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was taken up in dichloromethane and cooled to 0 ℃. Trifluoroacetic acid was added and the resulting solution was warmed to 23 ℃ over 2 hours. The solvent was then removed in vacuo and purified by reverse phase HPLC (gradient elution of 5-75% acetonitrile/water/0.1% trifluoroacetic acid for 20 min) to give compound 199(43mg, 54% yield) as a white solid (as TFA salt). ¹H-NMR(500MHz,MeOD)δppm 8.84(s,1H),8.31(d,1H),7.66(s,1H),7.34-7.30(m,1H),7.14-7.06(m,2H),6.98-6.95(m,2H),6.03(s,2H),4.58(t,2H),4.08(t,2H),3.90(s,2H)。

Compound I-200

To intermediate-20 (1 equivalent, the compound described in prior patent application publication WO 2014/144100), 2- (piperidin-1-yl) acetic acid (1.5 equivalents) and 4-dimethylaminopyridine (0.1 equivalent) in dichloromethane (13mL) was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.3 equivalents) in one portion at 0 ℃. The solution was immediately warmed to 23 ℃ and stirred for 15 hours. The solvent was removed in vacuo and purified by reverse phase HPLC (gradient elution of 5-75% acetonitrile/water/0.1% trifluoroacetic acid for 20 min) to give compound I-200(55mg, 69% yield) as a white solid (obtained as TFA salt).¹H-NMR(500MHz,MeOD)δppm 8.85(s,1H),8.30(d,1H),7.64(s,1H),7.35-7.31(m,1H),7.15-7.07(m,2H),7.00-6.98(m,2H),6.03(s,2H),4.59(t,2H),4.14(s,2H),4.09(t,2H),4.58(br s,2H),3.02(br s,2H),1.95-1.78(m,5H),1.52(br s,1H)。

Compound I-201

Intermediate-37 (1 equivalent, previously described in patent application publication WO 2014/144100) and sodium ethoxide (5 equivalents) were suspended in methanol at 150 ℃, added with one drop of water and microwaved for 30 minutes. The solvent was removed in vacuo and the residue diluted with saturated aqueous ammonium chloride and ethyl acetate (1:1 ratio). The layers were separated and the aqueous layer was extracted 2 times with ethyl acetate. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by reverse phase HPLC (gradient elution of 5-75% acetonitrile/water/0.1% trifluoroacetic acid for 20 min) gave compound 201(15mg, 20% yield) as a yellow solid.

¹H-NMR(500MHz,MeOD)δppm 7.84(s,1H),7.83(s,1H),7.35-7.31(m,1H),7.15-7.08(m,1H),6.95-6.92(m,1H),5.98(s,2H),2.60(s,3H)。

Compound 202

To a suspension of intermediate-20 (1 equivalent, described in the previous patent application WO 2014/144100), 4-dimethylaminopyridine (0.1 equivalent) and 1- (tert-butoxycarbonyl) piperidine-2-carboxylic acid (1.5 equivalent) in dichloromethane was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.8 equivalent) at 0 ℃. The solution was immediately warmed to 23 ℃ and stirred for a further 24 hours. The solution was poured into dichloromethane and 1N aqueous hydrochloric acid (2:1 ratio). The layers were separated and the organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was taken up in dichloromethane, cooled to 0 ℃ and trifluoroacetic acid (1/3 volumes of DCM) was added. The solution was stirred at 0 ℃ for 1 h, after which time the solvent was removed in vacuo and purified by reverse phase HPLC (gradient elution of 5-75% acetonitrile/water/0.1% trifluoroacetic acid for 20 min) to give the desired compound, compound 202(76mg, yield 97%) as a white solid (TFA salt form).¹H-NMR(500MHz,MeOD)δppm 8.86(s,1H),8.35(d,1H),7.68(s,1H),7.35-7.31(m,1H),7.15-7.07(m,2H),7.02-6.99(m,2H),6.04(s,2H),4.65-4.60(m,1H),4.58-4.53(m,1H),4.16-4.03(m,3H),3.43-2.99(m,1H),3.04-2.99(m,1H),2.29-2.26(m,1H),1.87-1.81(m,2H),1.76-1.57(m,3H)。

Compound 204

Sodium ethoxide (5.2 equivalents) and intermediate-38 (1 equivalent, described in the prior patent application publication WO 2014/144100) were suspended in methanol, added with a drop of water and treated with microwaves at 150 ℃ for 40 minutes. The solvent was removed in vacuo and the resulting residue was taken up in dichloromethane and water (1:1 ratio). The layers were separated and the aqueous layer was extracted 2 times with dichloromethane. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by reverse phase HPLC (5-75% acetonitrile/water/0.1% trifluoroacetic acid, gradient elution for 20 min) afforded the desired compound, compound 204(6mg, product yield) Rate 7%) as a white film.¹H-NMR(500MHz,MeOD)δppm 8.28(d,1H),7.78(s,1H),7.32-7.28(m,1H),7.14-7.10(m,1H),7.08-7.05(t,1H),6.86-6.83(t,1H),5.94(s,2H),4.48(q,2H),2.60(s,3H)。

Compound 205

N- (3- (dimethylamino) -2- (trifluoromethyl) allylidene) -N-methylmethylammonium hexafluorophosphate (2.4 equivalents), 1- (2-fluorobenzyl) -5- (isoxazol-3-yl) -1H-pyrazole-3-carboxamidine hydrochloride (1 equivalent, described in the prior patent application publications WO2013/101830 and WO 2014/144100) and triethylamine (2.4 equivalents) were suspended in acetonitrile and stirred at 23 ℃ for 3 hours. The reaction mixture was poured into water and dichloromethane (1:1 ratio). The layers were separated and the aqueous layer was extracted 2 times with dichloromethane. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (gradient elution of 0-50% ethyl acetate in hexanes) afforded the desired compound, compound 205(55mg, 91% yield) as a white solid.¹H-NMR(500MHz,CDCl₃)δppm 9.08(s,2H),8.51(s,1H),7.55(s,1H),7.25-7.21(m,1H),7.08-7.04(m,1H),7.00(t,1H),6.91-6.88(m,1H),6.64(s,1H),6.06(s,2H)。

Compound 206

A solution of picolinic acid (1.46 equivalents), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.3 equivalents), N-dimethylaminopyridine (0.1 equivalents) and intermediate-20 (1 equivalent, described in the prior patent application publication WO 2014/144100) in methylene chloride was stirred at room temperature for 20 hours. The solvent was removed in vacuo and the desired compound was purified by silica gel chromatography (gradient elution of 0-10% methanol in dichloromethane) as compound 206(59mg, 93% yield) as a white solid. ¹H-NMR(500MHz,CDCl₃)δppm 9.29(br s,1H),8.78(br s,1H),8.50(s,1H),8.37(br s,1H),8.24(br s,1H),7.49(br s,1H),7.43(br s,1H),7.24-7.21(m,1H),7.07-6.99(m,2H),6.91(br s,1H),6.66(s,1H),5.99(s,2H),4.71(br s,2H),4.16(br s,2H)。

Compound 217

The compound is prepared by the following steps:

step 1: synthesis of 2- (2-fluorophenyl) -N-hydroxyiminoamide

To a solution of hydroxylamine hydrochloride (2.2 equiv) and 2- (2-fluorophenyl) acetonitrile (1 equiv) in methanol and water (5:1 ratio) was added sodium bicarbonate (2.4 equiv). After stirring at 70 ℃ for 20 h, the methanol was removed in vacuo and the resulting solution was diluted with water and dichloromethane (1:2 ratio). The layers were separated and the organic layer was washed with saturated aqueous sodium chloride. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo to give the desired intermediate, 2- (2-fluorophenyl) -N-hydroxyiminoamide (1.13g, 90% yield) as a white solid.

Step 2: synthesis of ethyl 2- (2-fluorobenzyl) -1H-imidazole-5-carboxylate

A solution of ethyl propiolate (1.08 equiv) and 2- (2-fluorophenyl) -N-hydroxyiminoamide (1 equiv) in ethanol was heated at 80 ℃ for 5 hours at which time the solvent was removed in vacuo. The residue obtained is added to diphenyl ether and heated at 170 ℃ for 14 hours. The black reaction mixture was poured into hexane (4x volume of diphenyl ether) and stirred for 24 hours. The hexane was then decanted to give the crude product. Purification by silica gel chromatography (0-5% methanol in dichloromethane) gave the desired intermediate, ethyl 2- (2-fluorobenzyl) -1H-imidazole-5-carboxylate (1.8g, 27% yield) as a black solid.

Step 3: 2- (2-fluorobenzyl) -1-methyl-1H-imidazoleSynthesis of ethyl (4-carboxylate)

Ethyl 2- (2-fluorobenzyl) -1H-imidazole-5-carboxylate (1 eq) and cesium carbonate (1.2 eq) were suspended in acetonitrile and methyl iodide (1 eq) was added. After stirring at room temperature for 2 hours, the solvent was removed in vacuo and the black residue was diluted with dichloromethane and water (4:3 ratio). The layers were separated and the aqueous layer was extracted 2 times with dichloromethane. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (gradient elution with 0-80% ethyl acetate in hexanes) afforded the desired intermediate, ethyl 2- (2-fluorobenzyl) -1-methyl-1H-imidazole-4-carboxylate (375mg, 36% yield), as a black oil.

Step 4: synthesis of 2- (2-fluorobenzyl) -1-methyl-1H-imidazole-4-carboxamidine hydrochloride (intermediate-21)

Trimethylaluminum (5 equiv., 2N toluene solution) was added to a toluene suspension of ammonium chloride (5.5 equiv.) over 10 minutes. After stirring for 40 min, the solution was added to 2- (2-fluorobenzyl) -1-methyl-1H-imidazole-4-carboxylic acid ethyl ester (1 eq) and the resulting suspension was heated at 80 ℃ for 6H. The reaction mixture was cooled to 0 ℃ and methanol was added dropwise. The reaction mixture was warmed to 23 ℃ and stirred for an additional 1 hour. The resulting slurry was filtered through celite, then washed with 4:1 ether/methanol, then 1:1 dichloromethane/methanol to give the desired intermediate, 2- (2-fluorobenzyl) -1-methyl-1H-imidazole-4-carboxamidine hydrochloride (240mg, 73%) as a solid.

Step 5: synthesis of Compound 217

A solution of 1, 8-diazabicycloundecen-7-ene (1 eq), sodium 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (3.06 eq) and 2- (2-fluorobenzyl) -1-methyl-1H-imidazole-4-carboxamidine hydrochloride (1 eq)) in ethanol was heated in a sealed vial at 80 ℃ for 15H. The solvent was removed in vacuo, the residue suspended in methanol, the solid filtered off and the filtrate purified by reverse phase HPLC (5-75% acetonitrile/water/0.1% trifluoroacetic acid, gradient elution for 20 min) to give the desired compound (29mg, yield 63%) as a brown solid.¹H-NMR(400MHz,CD₃OD)δppm 8.17(d,1H),8.00(s,1H),7.42-7.37(m,1H),7.34-7.30(m,1H),7.24-7.16(m,2H),4.42(s,2H),3.79(s,3H)。

Compound 196

The compound is prepared by 5 steps:

step 1: synthesis of ethyl 1- (2-fluorobenzyl) -5-mercapto-1H-pyrazole-3-carboxylate

1- (2-Fluorobenzyl) -5-hydroxy-1H-pyrazole-3-carboxylic acid ethyl ester (1.8 equiv.) and Row's reagent (1 equiv.) were suspended in toluene (10mL) and heated at 120 ℃ for 2 hours. The solvent was removed in vacuo and the residue was taken up in dichloromethane. The resulting solid was filtered off and the filtrate was purified by silica gel chromatography (gradient elution with 0-50% ethyl acetate in hexanes) to give the desired intermediate, ethyl 1- (2-fluorobenzyl) -5-mercapto-1H-pyrazole-3-carboxylate (305mg, 55% yield) as an oil.

Step 2: synthesis of ethyl 1- (2-fluorobenzyl) -5- (methylthio) -1H-pyrazole-3-carboxylate

Potassium carbonate (2 equiv.) and ethyl 1- (2-fluorobenzyl) -5-mercapto-1H-pyrazole-3-carboxylate (1 equiv.) were suspended in tetrahydrofuran and methyl iodide (1 equiv.) was added. After 1.5 hours, the solution was diluted with ethyl acetate and water (1.5:1 ratio). The layers were separated and the aqueous layer was extracted 2 times with ethyl acetate. The organics were dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (gradient elution with 0-40% ethyl acetate in hexanes) afforded the desired intermediate, ethyl 1- (2-fluorobenzyl) -5- (methylthio) -1H-pyrazole-3-carboxylate (32mg, 61% yield), as an oil.

Step 3: 1- (2-Fluorobenzyl) -5- (methylsulfonyl) -1H-pyrazole-3-carboxylic acid ethyl ester

To a solution of A (1 equivalent) in dichloromethane was added 70% 3-chlorophenylperoxyacid (3 equivalents) at 0 ℃. The solution was immediately warmed to 23 ℃. After 3.5 hours, the reaction mixture was poured into ethyl acetate and saturated aqueous sodium bicarbonate. The layers were separated and the organic layer was washed with saturated aqueous sodium thiosulfate solution. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. Purification by silica gel chromatography (gradient elution with 0-40% ethyl acetate in hexanes) gave the desired intermediate, ethyl 1- (2-fluorobenzyl) -5- (methylsulfonyl) -1H-pyrazole-3-carboxylate (180mg, 89% yield), as a clear oil which solidified upon standing.

Step 4: 1- (2-Fluorobenzyl) -5- (methylsulfonyl) -1H-pyrazole-3-carboxamidine hydrochloride

To a toluene suspension of ammonium chloride (5.5 equivalents) was added a 2M toluene solution of trimethylaluminum (5.1 equivalents). After stirring for 30 min, the solution was bubbled through settling and added to 1- (2-fluorobenzyl) -5- (methylsulfonyl) -1H-pyrazole-3-carboxylic acid ethyl ester (1 eq). The resulting solution was stirred at 90 ℃ for 14 hours. The solution was cooled to 0 ℃ and methanol (5.5 eq) was added dropwise over 3 minutes. The suspension was then immediately warmed to 23 ℃ and stirred for 1 hour. After filtration of the suspension through celite, the filter cake was washed with 5mL 50:50 methanol/dichloromethane to give the desired intermediate, 1- (2-fluorobenzyl) -5- (methylsulfonyl) -1H-pyrazole-3-carboxamidine (52mg, 28% yield, HCl salt) as a white solid.

Step 5: synthesis of Compound 196

A solution of 1- (2-fluorobenzyl) -5- (methylsulfonyl) -1H-pyrazole-3-carboxamidine hydrochloride (1 eq), sodium 3-ethoxy-2-fluoro-3-oxoprop-1-en-1-ol (3.1 eq) and 1, 8-diazabicyclo [5.4.0] undec-7-ene (1 eq) in ethanol was heated at 90 ℃ for 4H. The solvent was removed in vacuo, the resulting residue was taken up in dichloromethane and the solid was filtered off. The filtrate was purified by silica gel chromatography (0-5% methanol in dichloromethane) to give the desired compound, compound 196(27mg, 47% yield) as a white solid.

¹H-NMR(500MHz,DMSO)δppm 13.42(br s,1H),8.15(br s,1H),7.60(s,1H),7.43-7.39(m,1H),7.29-7.25(m,1H),7.21-7.18(m,1H),7.14-7.12(m,1H),5.80(s,2H),3.41(s,3H)。

Compound I-188

A solution of tert-butyl 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (1 eq) and lithium tert-butoxide (2 eq) in dimethoxyethane (2ml) was stirred at 60 ℃ for 5 min. 4- (bromomethyl) -2-fluoro-1-methylbenzene (1.1 eq) was added thereto and the reaction was stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed under a stream of nitrogen. The resulting solid was dissolved in methanol (0.5ml) and concentrated aqueous HCl (140ul) and stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to give compound I-188(2.2mg, 6% yield) as a white solid.¹H NMR (500MHz, methanol-d 4) delta ppm 8.81(m,1H),8.06(m,1H),7.45(m,1H),7.17(m, 1H)),6.97(m,2H),6.91(m,1H),5.89(s,2H),2.22(m,3H)。

Compound I-208

This compound was synthesized using the same method as that used to synthesize compound I-188, except that 1- (bromomethyl) -2-fluoro-4-toluene was used as the alkylating agent to obtain the product (7.2mg, yield 30%) as a white solid.¹H NMR (500MHz, methanol-d 4) Δ ppm 8.79(s,1H),8.04(m,1H),7.43(s,1H),6.90(d,4H),5.93(s,2H),2.30(s, 3H).

Compound I-197

This compound was synthesized using the same method as that used to synthesize compound I-188, except that 1- (bromomethyl) -3-fluorobenzene was used as the alkylating agent to obtain the product (3.5mg, yield 15%) as a white solid. ¹H NMR (500MHz, methanol-d 4) Δ ppm 8.81(d,1H),8.04(d,1H),7.46(s,1H),7.32(d,1H),7.09(d,1H),7.02(m,2H),6.92(d,1H),5.94(s, 2H).

Compound I-213

This compound was synthesized using the same method as that used to synthesize compound I-188, except that 1- (bromomethyl) -4-fluorobenzene was used as an alkylating agent to obtain the product (2mg, yield 8%) as a white solid.¹H NMR (500MHz, methanol-d 4) Δ ppm 8.81(d,1H),8.03(d,1H),7.42(s,1H),7.34(m,2H),7.03(s,2H),6.90(m,1H),5.90(s, 2H).

Compound I-186

Use and combinationThis compound was synthesized in the same manner as in compound I-188, except that 4- (bromomethyl) -1-methyl-1H-pyrazole was used as the alkylating agent to give the product (6.5mg, yield 20%) as a white solid.¹H NMR(500MHz,DMSO-d6)δppm 13.12(m,1H),9.15(d,1H),8.12(br.s.,1H),7.70(s,1H),7.52(s,1H),7.45(s,1H),7.22(d,1H),5.65(s,2H),3.75(s,3H).

Compound I-212

This compound was synthesized using the same method as that used to synthesize compound I-188, except that 5- (bromomethyl) -1-methyl-1H-pyrazole was used as the alkylating agent to give the product (1.8mg, yield 6%) as a white solid.¹H NMR(500MHz,DMSO-d6)δppm 13.18(m,1H),9.15(d,1H),8.10(m,1H),7.58(s,1H),7.26(dd,2H),6.05(s,1H),5.90(s,2H),3.96(s,3H)。

Compound I-211

This compound was synthesized using the same method as that used to synthesize compound I-188, except that 5- (bromomethyl) isoxazole was used as the alkylating agent to give the product (5.1mg, yield 16%) as a white solid.¹H NMR(500MHz,DMSO-d6)δppm 13.16(m,1H),9.15(d,1H),8.53(d,1H),8.06(m,1H),7.61(s,1H),7.27(d,1H),6.41(s,1H),6.04(s,2H)。

Compound I-214

This compound was synthesized using the same method as compound I-188, except that 2,2, 2-trifluoroethyl trifluoromethanesulfonate was used as an alkylating agent to give the product (8.5mg, yield 27%) as a white solid. ¹H NMR (500MHz, methanol-d 4) Δ ppm 8.88(d,1H),8.07(d,1H),7.52(s,1H),7.02(d,1H),5.60(d, 2H).

Compound I-216

A solution of tert-butyl 3- (3- (5-fluoro-4-methoxypyrimidin-2-yl) -1H-pyrazol-5-yl) isoxazole (1 eq), triphenylphosphine (1.5 eq) and pyrimidin-5-ylcarbinol (1.5 eq) in dichloromethane/THF (1:1, 2ml) was cooled to 0 ℃. Diethyl azodicarboxylate (1.5 equivalents) was added thereto, and the reaction was allowed to warm to room temperature overnight. The reaction was concentrated under a stream of nitrogen and the methoxy intermediate was purified by reverse phase HPLC using a gradient of 30-80% acetonitrile in 0.1% formic acid. The desired fractions were concentrated to dryness and the resulting solid was dissolved in methanol (0.5ml) and concentrated aqueous HCl (140ul) and stirred at 60 ℃ overnight. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by reverse phase HPLC eluting with a gradient of 30-80% acetonitrile in 0.1% formic acid to give the product (1.9mg, 10% yield) as a white solid.¹H NMR (500MHz, methanol-d 4) Δ ppm 9.07(s,1H),8.83(m,3H),8.02(d,1H),7.46(s,1H),6.96(d,1H),5.98(s, 2H).

Compound I-215

This compound was synthesized using the same method as that used to synthesize compound I-216, except that pyrimidin-2-ylmethanol was used as the alkylating agent to obtain the product (1.4mg, yield 4.3%) as a white solid. ¹H NMR(500MHz,DMSO-d6)δppm 13.13(m,1H),9.04(d,1H),8.73(d,2H),8.07(m,1H),7.65(s,1H),7.41(t,1H),7.23(s,1H),6.07(s,2H)。

Compounds 191 and 192

Intermediate-39 (2.0g, 6.7mmol, obtained after hydrazine addition during the synthesis of intermediate-28), 2Diethyl 2-dimethyl-3-oxosuccinate (4.3g, 20.0mmol) and NaHCO₃A solution of (1.7g, 20.0mmol) in toluene (50mL) was heated at 100 deg.C overnight, then cooled to room temperature and filtered through celite. The filter cake was washed with ethyl acetate and the solvent was removed by rotary evaporation. Using SiO₂Purification, eluting with a hexane/ethyl acetate gradient, afforded the two positional isomer products as solids. Compound 191(0.82g, 27% yield).¹H-NMR(400MHz,CDCl₃) δ 10.95(br s,1H),8.56(d,1H),7.48(s,1H),7.3-7.4(m,1H),7.0-7.1(m,3H),6.65(d,1H),5.97(s,2H),4.18(q,2H),1.57(s,6H),1.22(t,3H) ppm compound 192(0.27g, 9% yield).¹H-NMR(400MHz,CDCl₃)δ11.05(br s,1H),8.52(d,1H),7.28(s,1H),7.2-7.3(m,1H),7.0-7.1(m,2H),6.94(t,1H),6.63(d,1H),6.00(s,2H),4.21(q,2H),1.66(s,6H),1.22(t,3H)ppm。

Compound 209

Step 1: compound 191(0.13g, 0.28mmol) was dissolved in phosphorus oxychloride (2.0mL, 21.5mmol) and the solution was heated at 105 ℃ for 1 hour. The solvent was removed in vacuo, the residue was dissolved in ethyl acetate, washed with 3X 5mL of water, brine, and then Na₂SO₄And (5) drying. After filtering off the drying agent, the crude product is passed through SiO₂Purification, eluting with a hexane/ethyl acetate gradient, gave the chlorinated intermediate as a white solid (100mg, 77% yield).

¹H-NMR(400MHz,CDCl3)δ8.52(d,1H),7.62(s,1H),7.2-7.3(m,2H),7.07(t,1H),6.64(d,1H),6.07(s,2H),4.22(q,2H),1.82(s,6H),1.23(t,3H)ppm。

Step 2: the chlorinated intermediate prepared in step 1 (100mg, 0.22mmol) was heated with ammonia in dioxane (0.5M, 10mL, 10mmol) in a sealed vial at 105 ℃ for 2.5 days. The mixture was cooled to room temperature, diluted with ethyl acetate (50mL), then washed with water (3X 5mL), brine, and Na₂SO₄And (5) drying. Filtering out the drying agent and dissolving the solutionConcentrating in air, and using SiO to obtain residue₂Purify by chromatography using a dichloromethane/ethyl acetate gradient as eluent. The product was obtained (19mg, yield 20%) as a white solid.¹H-NMR(400MHz,CDCl₃)δ8.47(s,1H),7.68(br s,1H),7.20(q,1H),7.04(t,1H),6.96(t,1H),6.76(t,1H),6.62(br s,1H),6.07(s,2H),6.02(br s,2H),4.17(q,2H),1.72(s,6H),1.20(t,3H)ppm。

Compound 195

Intermediate-36 (35mg, 0.09mmol), (R) -2- (aminomethyl) -3,3, 3-trifluoro-2-hydroxypropionamide (60mg, 0.35mmol) and N-ethyl-N-isopropylpropan-2-amine (0.10mL, 0.56mmol) were combined in dimethyl sulfoxide (1.5mL) and heated at 95 ℃ for 8 hours. The solution was cooled to room temperature, diluted with water (2mL) and the pH adjusted to 2-3 with 1N aqueous HCl. The solution was mixed with ethyl acetate (50mL), and the organic phase was washed with water (2X 5mL), brine, then Na₂SO₄Dried, filtered and concentrated by rotary evaporation. The residue was purified by preparative reverse phase HPLC eluting with a water/acetonitrile (0.1% trifluoroacetic acid) gradient to give the product as a white solid (11mg, 23% yield). ¹H-NMR(400MHz,CD₃OD) δ 8.83(br s,1H),8.27(br s,1H),7.49(br s,1H),6.9-7.0(m,2H),6.5-6.6(m,2H),5.86(s,2H),4.35(d,1H),4.16(d,1H) ppm note: exchangeable protons all appeared at 4.91ppm with a residual HOD peak.

Compound 194

Following general procedure B, the title compound was prepared from intermediate-1B and a dioxane solution of the reaction was heated at 90 ℃ for 5 hours using (S) -2-amino-3-hydroxypropionamide as the amine reactant. The crude product was purified by reverse phase HPLC to give the desired compound, (S) -2- ((2- (5-acetyl-1- (2-fluorobenzyl) -1H-pyrazol-3-yl) -5-fluoropyrimidin-4-yl) aminoYl) -3-hydroxypropionamide (8mg, 0.019mmol, 34% yield).¹H-NMR(500MHz,DMSO-d6)δppm 8.29(d,1H)7.71(s,1H)7.62(s,1H)7.30-7.36(m,1H)7.19-7.25(m,2H)7.11(t,1H)6.81(t,1H)5.83(s,2H)4.72(d,1H)3.77-3.86(m,2H),2.57(s,3H)。

Example 2A: determination of biological Activity Using LC/MS detection of the sGC-HEK-cGMP assay

Endogenous human embryonic kidney cells (HEK293) expressing soluble guanylate cyclase (sGC) were used to assess the activity of the test compounds. Compounds that stimulate sGC enzymes should cause an increase in the intracellular concentration of cGMP. HEK293 cells in poly-D-lysine-coated 384-well flat bottom plates at 1.5X 10⁴The density of cells/well was seeded in 50. mu.L volume of Darbeck modified eagle's medium supplemented with fetal bovine serum (10% final concentration) and penicillin (100U/mL)/streptomycin (100. mu.g/mL). Cells were incubated at 37 ℃ with 5% CO ₂Overnight in the humid chamber. The medium was aspirated and the cells were washed with 1-fold Hank's (Hank's) buffered saline solution (50 μ L). The cells were then incubated with 50. mu.L of 0.5mM 3-isobutyl-1-methylxanthine (IBMX) for 15 minutes at 37 ℃. Then, a test sample and a diethylenetriamine NONOate (DETA-NONOate) solution (the concentration of the test sample solution is x. mu.M, and the concentration of the DETA-NONOate solution is 10. mu.M; wherein x is one of the following concentrations) were added to the test mixture, and the resulting mixture was incubated at 37 ℃ for 20 minutes. After 20 min incubation, the test mixture was aspirated and 10% acetic acid containing 150ng/mL +3-cGMP (internal standard for LCMS) (50 μ L) was added to the cells. The plates were incubated in acetic acid solution at 4 ℃ for 30 minutes to stop the reaction and lyse the cells. The plates were then centrifuged at 1,000g for 3 minutes at 4 ℃ and the supernatant was transferred to a clean reaction plate for LCMS analysis.

30000nM
	7500nM
1875nM
	468.75nM
117.19nM
	29.29nM
7.32nM
	1.83nM
0.46nM
	0.114nM
0029nM

The cGMP concentration of each sample was measured using the following LCMS conditions (table 2) and a standard curve was calculated. The following final concentration of cGMP in ng/mL was utilized in 10% acetic acid with 150ng/mL +3cGMP (with isotope labeled cGMP 3 units higher than wild type): 1,5,10,50, 100, 250, 500, 1000, 2000 standard curves were prepared.

Table 2: LC/MS conditions, example 2

Data were normalized to the high contrast using the following equation: 100 (sample-low control)/(high control-low control), where low control is the average of 16 samples treated with 1% DMSO and high control is the average of 16 samples treated with 30 μ M of compound Y described below. Data were fitted with 4 parameters (log (agonist) contrast response-variable slope) using GraphPad Prism v.5 software, with n-2 fitting times for all compounds. The absolute EC50 was calculated from curve fitting and was defined as the concentration at which a given compound elicits a 50% high control reaction. Compounds that failed to elicit 50% of the minimal response were reported as >30 μ M. For compounds that are repeated or run with n higher than 2, the results presented herein are geometric averages of several results obtained. Table 3 summarizes the results obtained in this test for selected compounds of the invention.

TABLE 3A Whole cell Activity in HEK assay Using LC/MS (updated assay conditions, example 2A)

The code for the activity value of ((to)) sGC enzyme is defined as the absolute EC50, which is defined as the concentration at which a given compound causes a 50% high control reaction (compound Y). Compounds that failed to elicit 50% of the minimal response were reported as >30 μ M or ND. EC50Abs ≤ 100nM ═ a; 100nM < EC50Abs ≦ 1000nM ═ B; 1000nM < EC50Abs ═ C.

Example 2B: determination of biological Activity by cGMP-based GloSensor cell assay Using 384-well plates

Use of the expression GloSensor^TMHuman embryonic kidney cells (HEK293) of 40F cGMP (part number: CS182801, Promega) were used to evaluate the activity of the test compounds. Incorporation ofThe light-emitting biosensors (engineered luciferases) of these cells detect cGMP formed by compounds that stimulate sGC enzyme and emit fluorescence.

cGMP GloSensor cells were maintained in Darberg Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) (final 10%) and hygromycin (200 ug/ml). One day prior to the assay, cells were plated at 1.5X 10⁴Cell/well density was seeded in poly-D-lysine-coated 384-well leucogen-bottom-plate (Corning Cat No 35661) in 50 μ L volume of DMEM containing 10% FBS. Cells were incubated at 37 ℃ with 5% CO₂Overnight in the humid chamber. The following day, the medium was removed and used with 40. mu.l/well GloSensor^TMCells were replaced by 2mM (Promega Cat No E1291). The cells were treated at 25 ℃ for 90 minutes to allow the substrate to equilibrate in the cells. Test compounds and diethylene triamine NONOate (DETA-NONOate) in serum-free CO₂Dilutions were made to 3mM (20X) in independent medium and serially diluted at 4X dilutions to generate a 5X dose curve, and 10. mu.l of this solution was added to the wells (test compound solution concentration X. mu.M, DETA-NONONAte solution concentration 10. mu.M; where X is one of the following final concentrations).

30000nM
	7500nM
1875nM
	468.75nM
117.19nM
	29.29nM
7.32nM
	1.83nM
0.46nM
	0.114nM
0.029nM

For kinetic studies, fluorescence was measured immediately at 0.2 sec/well using Envision (Perkin Elmer model). For the endpoint SAR screening, data were collected after 55 minutes incubation at room temperature.

Data analysis was performed as described above in example 2A.

Table 3B whole cell activity was measured by GloSensor-based cell assay in 384-well plates (example 2B).

The code for the activity value of ((to)) sGC enzyme is defined as the absolute EC50, which is defined as the concentration at which a given compound causes a 50% high control reaction (compound Y). Compounds that failed to elicit 50% of the minimal response were reported as >30 μ M or ND. EC50Abs ≤ 100nM ═ a; 100nM < EC50Abs ≦ 1000nM ═ B; 1000nM < EC50Abs ═ C.

Example 3A: determination of biological Activity by thoracic aortic Ring test

The thoracic aortic annulus was dissected from male Sprague-Dawley rats weighing 275-. Tissue was immediately transferred to a tissue that had been treated with 95% O₂And 5% CO₂Air-filled for 30 minutes into an ice-cold Krebs-Hansleit (Krebs-Henseleit) solution. After removal of connective tissue, the aortic section was cut into 4 rings (-2 mm each) and hung on 2L-shaped hooks, one of whichFixed to the bottom of a tissue bath (scohler organ bath, harvard instrument), and the other connected to a force sensor (F30 force sensor, harvard instrument). The bath containing Krebs Henseleit solution (10mL) was heated to 37 ℃ with 95% O ₂And 5% CO₂And (6) inflating. The ring was brought to an initial tension of 0.3-0.5g and gradually increased to a static tension of 1.0g over 60 minutes. With Krebs Henseleit solution (heated to 37 ℃ and 95% O)₂And 5% CO₂Air-filled) rinse the ring at 15 minute intervals until a stable baseline was obtained. After maintaining a static tension of 1.0g (about 10 minutes) without adjustment, the loop was considered stable. The ring was then contracted with 100ng/mL phenylephrine by adding 100. mu.l of a 10. mu.g/mL phenylephrine stock solution. Tissues that achieved stable contraction were then treated in an accumulative, dose-dependent manner with test compounds prepared in dimethyl sulfoxide (DMSO). In some cases, Krebs-Henseleit's solution (heated to 37 ℃ and 95% O)₂And 5% CO₂Air-filled) tissue was rinsed three times over a 5 minute period to stabilize at baseline and then used to characterize other test samples or DMSO effects. All data were collected using HSE-ACAD software provided by the haver instrument. Percent relaxation effect was calculated in Microsoft Excel using the reported strain values for 100ng/mL phenylephrine treatment as 0% inhibition and 100. mu.M 3-isobutyl-1-methylxanthine treatment as 100% inhibition. EC50 values were calculated using concentration-response curves generated by GraphPad Prism software.

Example 3B: determination of biological Activity by thoracic aortic Ring test Using alternative methods

As an alternative thoracic aortic ring test, the procedure of example 3 was used except that the percent relaxation effect was calculated in Microsoft Excel using the recorded 100ng/mL phenylephrine treatment as 0% inhibition and the original resting tension of the tissue as the tension value of 100% inhibition after rinsing the tissue with buffer.

Example 4: blood pressure changes in Sprague-Dawley rats

Male rats (250-350g body weight, provided by haren laboratories) were anesthetized with ketamine/xylazine and heparinized saline filled into a catheter implanted in the right femoral artery. The catheters were placed externally between the scapulae, capped, and the animals were allowed to recover post-surgery for at least 7 days before any compound testing. Prior to testing, animals were maintained on a normal diet and were free to drink drinking water under a 12 hour light-dark cycle.

On the day of the experiment, the catheter was uncapped and connected to a tether (Instech lab) and pressure sensor (haver instrument) under isoflurane inhalation anesthesia. Blood pressure and heart rate were then captured and analyzed with a dedicated data capture system (PowerLab, AD instrument). The data sampling rate is set to 1 cycle per second. Once connected, each rat was allowed to recover from anesthesia and baseline blood pressure and heart rate levels were established in these conscious, freely moving animals. Once baseline was established, vehicle (0.5% methylcellulose or 100% PEG400) or test samples were administered orally (PO, 10mg/kg) and the effect on blood pressure and heart rate was monitored for 24 hours.

Data are reported as hourly averages and blood pressure changes are calculated from hourly subtraction of individual baselines (Table 4)

TABLE 4

@ code definition for mean arterial peak change in rats in baseline at 10 mpk:

peak change <0 in baseline at-10 <3 or 10mpk

A peak change in the baseline of-10 or less at-20 or less 3 or 10mpk

Peak change < -20 in baseline at C-3 or 10mpk

Example 5: purified human recombinant sGC alpha 1 beta 1 enzyme assay in the presence of diethylenetriamine NONAte (DETA-NONONAte) (nitric oxide donor)

Purified human recombinant soluble guanylate cyclase α 1 β 1(h sGC) from Enzo Life sciences (P/N: ALX-201-177) was used to evaluate the activity of test compounds. The test reaction contained 0.1M Tris (pH8.0), 0.5mg/mL BSA, 2mM DTT, 4mM MgCl₂30 μ M DETA NONAte (Enzo Life sciences P/N: ALX-430-014) and 12.5ng/ml human soluble guanylate cyclase. Test compounds in DMSO were then added (3-fold titration of compounds on a 10-point curve starting at 30uM final concentration, all samples having 3% DMSO final concentration). Guanosine 5' -triphosphate (Sigma Aldrich) P/N: G8877 was added to a final concentration of 300. mu.M and the enzyme reaction was incubated at 37 ℃ for 20 minutes (100. mu.L, 384 well plate format). Controls contained either 3% DMSO (low control) or 30uM compound Y (high control). After 20 min incubation, the reaction was stopped by adding 100 μ L of ice-cold 20% acetic acid.

The cGMP concentration in all samples was determined using the cGMP HTRF (Cisbio P/N: 62GM2PEC) assay according to the manufacturer's instructions. cGMP standard curves were fitted using GraphPad Prism v.6 software using a 4-parameter fit (log (inhibitor) versus response-variable slope). The samples were diluted appropriately to ensure that the values fell within the linear range of the standard curve.

Data were fitted using GraphPad Prism v.6 software using a 4-parameter fit (log (agonist) contrast response-variable slope). Calculating EC in Curve fitting₅₀And combining EC₅₀Defined as the concentration at which the compound elicits 50% of the maximal response of 30 μ M compound Y (high control compound).

TABLE 5 enzyme data

EC50Abs<100nM＝A

≤100nM<EC50Abs<1000nM＝B

≤1000nM＝C

Example 6: description of animal models:

lamb model of pulmonary hemodynamics using inhaled sGC stimulators

("inhaled soluble guanylate cyclase agonist induces selective pulmonary vasodilation," Oleg V. et al, J.Med.Med.Res. and intensive Care, Vol.176, 2007, p.1138)

Whether the inhaled novel dry powder microparticle formulation containing sGC stimulator will produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension can be tested by the method disclosed below. The co-administration of microparticles of sGC stimulator and Inhaled Nitric Oxide (iNO) in this system can also be evaluated. Finally, it can be examined whether microparticles inhaled with sGC stimulators produce pulmonary vasodilation when the response to iNO (inducible nitric oxide synthase) is impaired.

The scheme is as follows: in awake, spontaneously breathing lambs with vascular and tracheostomy tube devices, U-46619 was intravenously infused to increase the mean pulmonary artery pressure to 35mm Hg. Inhalation of microparticles consisting of BAY 41-2272, BAY41-8543 or BAY58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased pulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY41-8543 or BAY58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO increases following inhalation of BAY41-8543 microparticles. Intravenous administration of 1H- [1,2,4] oxadiazolo [4,3-a ] quinoxalin-1-One (ODQ) to repair the heme group of oxidized sGC significantly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and pulmonary cGMP release induced by inhalation of BAY58-2667 microparticles was greatly enhanced following treatment with ODQ. Therefore, inhalation of microparticles containing sGC agonists can provide an effective new treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is oxidatively impaired by sGC. Note that: BAY 41-2272, BAY41-8543 are sGC stimulators, and BAY58-2667 are sGC activators.

In vitro (ex vivo) model of electric field-stimulated guinea pig tracheal smooth muscle for assessment of bronchiectasis

The bronchodilatory effect of sGC stimulators can be assessed by using the following system. This system allows us to determine the efficacy, efficacy and duration of action of several sGC stimulators, as well as to assess potential side effects, such as blood pressure or heart rate changes.

Animals: guinea pig, Gonghara (Dunkin Hartley), male, was cultured in complete barrier and confirmed to receive 525-. Guinea pigs were housed in controlled environments (air flow, temperature and humidity) with a gold foil mattress with a solid chassis of gold foil in each group. Food (FD1, special dietary service) and water were provided ad libitum.

Guinea pig tracheal smooth muscle contraction response to EFS. Evaluation of compound potency and efficacy:

on each experimental day, by exposure to elevated concentrations of CO₂To kill the guinea pigs and remove the trachea. The trachea is cleared of external tissue and cut longitudinally on a line opposite the muscle, opened and cut to 2-3 cartilaginous ring widths. A cotton ring is attached to one end of each trachea strip and a length of cotton is attached to the other end. The tracheal strip was then suspended between two platinum electrodes in the Myobath system (world precision instruments stevensi, UK) using a tissue holder. The loop is attached to a hook at the bottom of the tissue holder and the other end is attached to the arm of a FORT10 force sensor (UK, world precision instruments stevensi), ensuring that the tissue is located between the two platinum electrodes. The entire assembly was then placed from overhead at 37 ℃ into a 10ml tissue bath containing modified Kreb's-Henseleit buffer bubbled with high oxygen. Each piece of tissue was subjected to 1g of tension, washed, and then allowed to stabilize for 1 hour. Once the tissue stabilized, the device for electric field stimulation was set to deliver stimulation at a frequency of 80Hz with a pulse width of 0.1ms with gated unipolar pulses every 2 minutes using a DS80008 channel digital stimulator (world precision instruments stevensi, UK). Voltage response curves were performed at 2,4,6,7,8,10,12V for each tracheal strip, and then the next maximum voltage was selected to be applied to each tissue during the remainder of the experiment. Guinea Pig Tracheal Smooth Muscle (GPTSM) contraction was induced using sub-maximal Electric Field Stimulation (EFS) (contraction could also be induced by the use of spasmodic substances such as methacholine or histamine as described in Coleman et al). The compound is added at 3X 10 ^-2M was dissolved in 100% DMSO and stored in aliquots at-20 ℃. Separate aliquots were used for each experiment. Tissues were washed with Kreb's buffer and stimulated for 1 hour using the previously determined sub-maximal voltage to establish a stable baseline contraction prior to assessing compound activity.

A cumulative Dose Response Curve (DRC) determination was then performed for each test substance and the changes in smooth muscle contraction were measured. The effect of each test substance in each experiment was expressed as a percent inhibition of baseline contraction, normalized to the relevant vehicle control. Three experiments were performed using tissues from three different animals. Data from all three experiments were pooled, DRCs plotted, and potency and efficacy of the test substances determined. The potency of ipratropium bromide was evaluated with test compounds, and in agreement with data previously generated in this system, the IC50 was determined to be 0.86nM (95% Cl, 0.78-0.94).

A novel multifunctional enema system for assessing some spasms and relaxants of tracheal smooth muscle using guinea pig isolation, r.a. coleman et al, journal of pharmacological methods, 21, 71-86, 1989.

Mouse models of diseases involving alterations in CFTR functional factors

These diseases include cystic fibrosis, pancreatic disease, gastrointestinal disease, liver disease, cystic fibrosis related diabetes (CFRO), dry eye, dry mouth and xerosis (Sjoegren) syndrome.

By using transgenic mice expressing or not expressing the af 508CFTR channel, differences in nasal potential differences and salivary secretion in the presence of test sGC stimulators can be measured by using the following literature protocol (see WO 2011095534).

Salivary secretion test in Delta (.6.)50S-CFTR mice

A.6.50S-CFTR (original FVB genetic background backcross from university of rhadand Islams at 10-14 weeks of age, 1S-36g in weight, both sexes over 12 generations) was used in the trial, homozygous heterozygous for 15 male and female. Vardenafil solutions were prepared in sterile saline at concentrations of 0.07,0.14 and 0.42mg/kg BW, while the sGC stimulators BAY41-2272 were dissolved in 0.01,0.03,0.1 and 0.3mg/kg BW containing 50% ddH₂O, 40% PEG400 (polyethylene glycol 400) and 10% ethanol. The mice were administered the substance or a suitable vehicle by intraperitoneal injection (5ml/kg BW) 60 minutes prior to the salivary secretion assay. After 60 minutes, mice were anesthetized with a combination of 25-bronchial ketamine and diazepam. An 8 ml sterile saline solution containing 1 ml of 5mg/ml diazepam and 1 ml of 100mg/ml ketamine was prepared. Through the peritoneumAnesthesia was induced by internal injection of solution (10ml/kg BW). After anesthesia, mice were pretreated by subcutaneous injection of 1mM atropine (501-11) into the left cheek to avoid cross-stimulation of cholinergic receptors. A small strip of whatman filter 5 paper was placed in the previously injected cheek for 4 minutes to absorb any saliva secreted after the injection of atropine. The first piece of filter paper was removed and replaced with a second piece of pre-weighed filter paper. Thereafter, 501-11 containing a solution of 100I-IM isoproterenol and 1mM atropine was injected into the left cheek at the same site to induce salivation by the adrenergic mechanism. The time of 10 isoproterenol injections was taken as time zero and the filter paper strips were replaced every 10 minutes for a total collection time of 30 minutes. Each piece of filter paper was immediately placed and sealed in a pre-weighed vial. After all samples were collected, each vial was re-measured and the weight of all samples was recorded. The difference in total vial-plus-paper weight measured before and after collection of saliva 15 was taken as the net weight of saliva secreted during collection. The total amount of salivary secretion was calculated as the weight of saliva divided by the number of minutes required for each collection and then normalized to the mass of the mouse (grams). Results are expressed as mean percentage increase in n mice compared to placebo treatment. The analysis was performed statistically after one-way ANOVA test 20, followed by post hoc Bonferoni (Bonferoni) analysis; indicates statistical significance, p value <0.05/<0.01/0.001, n.s. is not significant.

These animal studies were performed with a number of sGC stimulators, sGC activators and PDE5 inhibitors. The results indicate that the compounds of the invention are useful in the treatment of cystic fibrosis, pancreatic disorders, gastrointestinal disorders, liver disorders, cystic fibrosis related diabetes (CFRO), dry eye, dry mouth and xerosis (Sjoegren) syndrome.

Neuromuscular diseases

It has previously been shown that a mislocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the sarcoplasmic muscle is observed in a wide range of non-nutritive neuromuscular conditions associated with impaired motor status and catabolic stress. Assessing electromyographic localization of nNOS is one tool for assessing muscle biopsies of patients in various forms of genetic and acquired neuromuscular diseases. The nNOS level of the sarcolemma is related to mobility and functional status.

Similar assessments can be used to determine nNOS localization in animal models of non-nutritive myopathy, following the literature protocol ("myofascial nNOS lesions common in acquired and inherited neuromuscular diseases"); e.l. finnger Hedderick et al, neurology, 2011, 76(11), 960-.

nNOS mislocalization in mouse model of acquired muscle atrophy

Two mouse models showing muscle atrophy without impaired mobility are described below: high dose corticosteroid treatment and short term hunger. Mice treated with steroids or starvation for 48 hours showed a significant decrease in total body mass and normalized wet skeletal muscle mass. Morphological analysis of skeletal muscle specimens of both models showed muscle atrophy (defined as a significant reduction in mean minimum Feret fiber diameter (n-5 per group) compared to age matched controls). Immunofluorescent staining of dystrophin, alpha-sarsan protein and alpha-1-synthetic protein revealed a normal dystrophin localization, indicating integrity of the DGC complex. However, both steroid-treated and starved mice showed no or severely reduced myomembrane nNOS staining. Real-time PCR of NOS family proteins (nNOS, eNOS, iNOS) showed no significant difference in the expression level of any of the 3 transcripts in steroid-treated mice (n ═ 8 per group). Furthermore, Western blot analysis of nNOS, iNOS and eNOS showed no difference at the protein level.

These murine models can be used to assess the effect of sGC stimulators (e.g., of the present invention) on muscle wasting symptoms and related disease states.

Starved mice showed a 1-fold reduction in nNOS and iNOS transcript expression compared to wild-type mice (control group n ═ 9, starved group n ═ 7). However, the protein levels of nNOS, iNOS and eNOS showed no difference between the control group and the starved mice (n ═ 4 per group). These data indicate that abnormal localization of nNOS occurs in mice with severe muscle atrophy, although overall mobility remains unchanged, supporting the concept that in addition to impaired mobility, other triggers such as catabolic stress may be associated with loss of the nNOS' sarcolemma.

Maintenance of skeletal muscle nNOS localization during hibernation (with squirrel study)

Skeletal muscle specimens from hibernating 13-row squirrels have been used to assess the effect of immobility and catabolic stress on nNOS localization in the context of maintaining muscle homeostasis and integrity. These animals are hibernating mammals that protect from skeletal muscle atrophy during hibernation. Although dormant for 5 months, almost completely motionless, with no caloric intake, the myofascial expression of nNOS was retained. These data, together with patient and mouse data, suggest that biochemical control of nNOS localization is complex and, importantly, that retained myofascial nNOS may be important in maintaining muscle homeostasis.

These results also indicate that targeting aberrant NO signaling (e.g., with sGC stimulators such as those described herein) may prove beneficial to a wide range of neuromuscular disease patients.

Mouse model of muscular dystrophy (BMD and DMD)

Becker Muscular Dystrophy (BMD), which is characterized by progressive skeletal muscle wasting, is caused by mutations in the muscle protein dystrophin. In human studies, Martin et al (see "tadalafil relief of muscle ischemia in patients with becker muscular dystrophy"; Elizabeth a. Martin et al, journal of scientific transformation medicine, 4,162, 155 (2012); vascular targeted treatment of duchenne muscular dystrophy "; Ennen et al, skeletal muscle, 2013, 3: 9) evaluated the attenuation of muscle reflex sympathetic vasoconstriction in 10 BMD patients induced by exercise and 7 age-matched healthy male controls. This is a protective mechanism that optimizes skeletal muscle perfusion to meet the motor metabolic demands. Reflex vasoconstriction is caused by simulated upright stress and is measured in the form of a rhythmic handle by forearm muscle rest or mild exercise. First, researchers showed that 9 of 10 BMD patients were deficient in motor-induced reduction of reflex vasoconstriction, with the common dystrophin mutation disrupting neuronal NO synthase (nNOS) targeting to the muscle sarcolemma. Then, in a double-blind randomized placebo-controlled crossover trial, the authors showed that 8 out of 9 patients restored normal blood flow regulation by a single oral dose of 20mg tadalafil (a specific PDE5 inhibitor).

The effect of drugs acting on the NO pathway can be assessed by using a duchenne muscular dystrophy protein-deficient mdx mouse model associated with DMD. The model also shows that inhibitors of phosphodiesterase 5(PDE5) alleviate some of the features of the dystrophic phenotype, including vasospasm of skeletal muscle microvasculature which can lead to muscle damage and fatigue.

With the movement of healthy skeletal muscle, myofascial nNOS-derived NO relieves local alpha-adrenergic vasoconstriction, thereby optimizing perfusion to meet the metabolic demand of the active muscle. In mdx mice (models of BMD and DMD), nNOS null mice and boys with DMD causing functional muscle ischemia, this protective mechanism (called functional sympathetic) is lost. Repeated episodes of functional ischemia can accelerate use-dependent injury to muscle fibers that have been weakened due to dystrophin deficiency.

In mdx mice, many of the characteristics of the dystrophic phenotype can be improved by a variety of strategies for enhancing NO signaling, including transgene expression of nNOS, transgene expression of dystrophin minigene to restore myofascial nNOS (and thus functional sympathetic nerve), administration of the NOs substrate L-arginine (24,25), treatment with NO donor drugs, and inhibition of phosphodiesterase 5A (PDE5A) by the drugs tadalafil or sildenafil. After brief exercise, these PDE5A inhibitors that extend the half-life of inosine 3', 5' -monophosphate (cGMP), a downstream target of NO in vascular smooth muscle, have been shown to reduce muscle ischemia as well as injury and fatigue in mdx mice. In addition, these drugs showed improvement in cardiac dynamics in mdx mice and nourished dystrophic skeletal muscle and prolonged survival of dystrophin deficient zebrafish.

These findings support the important role of myomembranous nNOS in the modulation of sympathetic vasoconstriction in human skeletal muscle, and suggest that targeting aberrant NO pathways (e.g., by using sGC stimulators of the invention) may be a useful therapeutic approach for the treatment of human BMD and DMD.

Sickle cell disease

Sickle Cell Disease (SCD) or Sickle Cell Anemia (SCA) or recurrent polycythemia is an inherited blood disorder characterized by red blood cells that exhibit an abnormal, rigid sickle shape. Sickling reduces the flexibility of the cell and leads to the risk of various complications. Sickling occurs due to a mutation in the hemoglobin gene. Individuals with one copy of the failure gene can display both normal and abnormal hemoglobin phenotypes. This is an example of co-dominance. In 1994, the average life expectancy of patients with this condition in the united states was 42 years in men and 48 years in women, but today patients can live up to 70 years due to better disease management.

Sickle cell anemia is a form of sickle cell disease in which the mutation causing HbS is homozygous. Sickle cell anemia is also known as "HbSS", "SS disease", "hemoglobin S" or an array of these names. In heterozygotes, i.e., those having a sickle gene and a normal adult hemoglobin gene, this condition is referred to as "HbAS" or "sickle cell signature". Other rare sickle cell diseases are complex heterozygous states, with the patient's HbS being caused by a mutation in only one copy, and another aberrant hemoglobin allele being present in the other copy. They include sickle hemoglobin C disease (HbSC), sickle beta-plus-thalassemia (HbS/beta) ⁺) And sickle-0-thalassemia (HbS/beta)⁰)。

Although the sickle cell disease is central to the pathophysiology of Red Blood Cell (RBC) sickle-like and rheological abnormalities, vascular dysfunction caused by the complex interaction between sickle red blood cells (sRBC), endothelial cells, platelets and leukocytes plays an equally important role. In sickle cell disease, endothelial activation is associated with sickle cell-mediated events of hypoxia reperfusion (see, e.g., "progress in understanding the pathogenesis of cerebrovascular vasculopathy in sickle cell anemia", p. connes et al, journal of hematology, uk, 2013, 161, 484-98). Sickle cells and adhesion to the endothelium initiate vascular occlusion by impairing blood flow. Subsequent proliferation of inflammatory mediators and endothelial activation trigger a series of events that lead to vascular injury. The pathophysiological responses from intermittent hypoxic reperfusion of these vaso-occlusive events are characterized by increased production of cytokines, leukocyte upregulation and activation of procoagulants and adhesion molecules, and simultaneous inhibition of cytoprotective mediators.

Leukocytosis is associated with almost every manifestation of sickle cell disease, emphasizing the role of inflammation in the pathophysiology of sickle vascular disease. Even at baseline levels, sickle cell disease patients develop elevated levels of proinflammatory cytokines including C-reactive protein (CRP), Tumor Necrosis Factor (TNF), interleukin-1 (IL-1), and interleukin-8 (IL-8). In vitro studies have shown that sRBC promotes endothelial upregulation of TNF- α and IL-1- β (8-10). Microarray studies of activated monocytes have shown differential expression of genes involved in inflammation, heme metabolism, cell cycle regulation, antioxidant responses and angiogenesis. Recently, differential expression of activated B cells (NF κ B/p65), the nuclear factor κ -light chain enhancer of Kruppel-like factor 2(KLF2), and other transcription factors that regulate inflammatory pathways in infants with sickle cell disease have been shown to increase stroke risk.

In transgenic mouse models (see "novel therapies targeting endothelial cells in sickle cell disease", c.c. hoppe, hemoglobin, 35 (5-6): 530-. Oxidative stress occurs through the formation of Reactive Oxygen Species (ROS). Consumption of NO occurs through hemoglobin (Hb) -mediated clearance, ROS consumption, and arginase-mediated substrate consumption. In sickle cell disease, the clearance system that normally removes circulating free Hb is saturated. Free Hb consumes NO, resulting in endothelial dysfunction. Thus, the normal balance of vasoconstriction and vasodilation is predisposed to vasoconstriction, endothelial activation, oxidative stress and proliferative vasculopathy.

Therapies directed at restoring NO homeostasis have shown promise in preliminary studies on sickle cell disease patients. Previous in vitro studies and studies of other patient populations have shown NO-mediated down-regulation of endothelial adhesion molecule expression. Based on these observations, the use of inhaled NO was studied in sickle cell patients presenting VOE and found a related trend of reduced pain scores, reduced analgesic requirements and reduced hospital stays.

These findings were reproduced in a recent randomized placebo-controlled trial evaluating acute VOE of inhaled NO for treatment of sickle cell disease adult patients, and the results showed that inhaled NO significantly reduced pain scores compared to placebo and correlated with a trend towards reduced parenteral morphine use. The results of a complete phase II trial from an adult sickle cell disease patient treated with acute VOE inhaled NO have not been provided (clinical trial, government number NCT 00023296). Another phase II trial of inhaled NO for VOE treatment in sickle cell disease children will be completed (U.S. dental and craniofacial institute, government number NCT 00094887). The potential therapeutic effect of inhaled NO on ACS in sickle cell disease is currently being evaluated by two independent phase II/III trials in france for children and adults comparing the efficacy of inhaled NO in infants with placebo or standard care in ACS patients (U.S. dental and craniofacial institute, government numbers NCT01089439 and NCT 00748423).

Dietary supplementation of the NO synthase substrate L-arginine has been extensively studied in sickle cell disease as a means of increasing NO bioavailability. In sickle mice, high doses of oral L-arginine have been shown to reduce the effects of gaados (Gardos) channel activity, compact cell formation and hemolysis, and functional improvement of vascular reactivity.

Sildenafil, an agent intended to amplify the action of endogenous NO by inhibiting PDE5, a downstream mediator of NO, is widely used in the general population to treat primary PHT. Preliminary studies in sickle cell disease patients for severe PHT reported improvements in PAP and exercise capacity following treatment with sildenafil. A multicenter trial to test the safety and efficacy of sildenafil in sickle cell disease patients with doppler-defined PHT (sildenafil treatment for pulmonary hypertension and sickle cell disease, Walk-PHaSST) was prematurely discontinued due to higher frequency and severe side effects including increased rates of VOE, headache and vision impairment in the treatment group.

The direct NO donor properties of nitrite and niacin were also investigated. In the experimental phase I/II clinical trial, sodium nitrite infusion in adult sickle cell disease patients enhanced forearm blood flow consistent with the NO donor mechanism of action. Large phase I/II trials are now underway to investigate whether nitrite infusion as an adjunctive therapy during acute VOEs will improve microvascular blood flow and tissue oxygenation (american dental and craniofacial institute, government number NCT 01033227). The effect of niacin on the improvement of endothelium-dependent vasodilation was also evaluated in a phase II randomized control trial (american dental and craniofacial institute, government number NCT 00508989).

The above results indicate that targeting an aberrant NO pathway in a cellular disease (e.g. by using sGC stimulators of the invention) may be an effective therapeutic approach to treat the disease. In blood, 2001, 98(5), 1577-84; journal of clinical research, 2004,114(8), 1136-45; and uk journal of hematology 2004,124(3), 391-402 describe murine models of sickle cell anemia that can be used to assess the role of sGC stimulators, such as the sGC stimulators of the invention, in this disease state.

Bladder dysfunction

Studies have shown that sGC activator BAY 60-2770 improves bladder overactivity in obese mice (see "soluble guanylate cyclase activator BAY 60-2770 improves obesity mouse bladder overactivity", Luiz O Leiria et al, J.Urology, 2013, doi: 10.1016/j.juro.2013.09.020.). The animal models described in this publication can similarly be used to assess the effect of sGC stimulators (e.g., the sGC stimulators of the invention) on bladder overactivity.

The same group of researchers also described a rat model of bladder dysfunction (NO-effective rats, F Z monaca et al, neurology and urodynamics, 30,456-60, 2011), and demonstrated the protective role of BAY-2272(sGC activator) in this model. The animal models described in this publication can similarly be used to assess the effect of sGC stimulators (e.g., sGC stimulators of the invention) on bladder dysfunction associated with detrusor smooth muscle overactivity.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" (and any form of comprising, such as "comprises" and "comprising"), "has" (and any form of having, such as "has" and "having"), "includes" (and any form of including, such as "includes" and "including") and any other grammatical variants thereof are open linking verbs. As a result, a method or apparatus that "comprises," "has," "includes" or "contains" one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements. Similarly, a step of a method or an element of a device that "comprises," "has," "includes" or "contains" one or more features possesses those one or more features, but is not limited to possessing only those one or more features. Further, a device or structure configured in a certain way is configured in at least that way, but may also be configured in ways not listed.

As used herein, the terms "comprising," "having," "including," "containing," and other grammatical variations include the terms "consisting of and" consisting essentially of.

The phrase "consisting essentially of," or grammatical variants thereof, as used herein is intended to specify the presence of stated features, integers, steps or components but does not preclude the addition of one or more additional features, integers, steps, components or groups thereof, but only so far as the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

All publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as though fully set forth.

Subject matter incorporated by reference is not to be considered a replacement for any claim limitation unless explicitly stated otherwise.

Where reference is made throughout this specification to one or more ranges, each range is intended as a shorthand format for presenting information, wherein the range is understood to include each discrete point within the range, as if fully set forth herein.

Although several aspects and embodiments of the present invention have been described and depicted herein, those skilled in the art may affect alternative aspects and embodiments to achieve the same objectives. Accordingly, the invention and the appended claims are intended to cover all such further and alternative aspects and embodiments that fall within the true spirit and scope of the invention.

Claims (2)

1. A compound shown in table A, B, C below or table D below, or a pharmaceutically acceptable salt thereof:

TABLE A

TABLE B

Watch C

Table D

。

2. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and one or more excipients.